Anti-CD19/anti-CD3 bispecific antibody, T cells secreting the same, method of preparation and use thereof

Abstract

The present invention relates to a bispecific antibody comprising an anti-CD19 single-chain fragment variable and an anti-CD3 single-chain fragment variable. The present invention also relates to T cells secreting the bispecific antibody, method of preparation of T cells secreting the bispecific antibody and uses thereof in the treatment of a hematological malignancy selected from the group consisting of lymphoma, leukemia and myeloma.

Claims

1. T cells characterized by secreting a bispecific antibody comprising: an anti-CD19 single-chain fragment variable (scFv) comprising an anti-CD19 light chain variable domain V.sub.LCD19 and an anti-CD19 heavy chain variable domain V.sub.HCD19, an anti-CD3 single-chain fragment variable (scFv) comprising an anti-CD3 heavy chain domain V.sub.HCD3 and an anti-CD3 light chain domain V.sub.LCD3, wherein the domains are arranged in the order V.sub.LCD19-V.sub.HCD19-V.sub.HCD3-V.sub.LCD3, wherein said bispecific antibody comprises: an anti-CD19 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 10, an anti-CD19 light chain CDR2 region consisting of the amino acid sequence Ile-Ala-Ser, an anti-CD19 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 11, an anti-CD19 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 12, an anti-CD19 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 13, an anti-CD19 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 14, an anti-CD3 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 15, an anti-CD3 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 16 and an anti-CD3 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: an anti-CD3 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 18, an anti-CD3 light chain CDR2 region consisting of the amino acid sequence Asp-Thr-Ser, an anti-CD3 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 19, wherein the bispecific antibody is expressed in the cells from a polynucleotide encoding the bispecific antibody and a signal peptide selected from a human kappa light chain signal peptide or a human lambda light chain signal peptide.

2. The T cells according to claim 1 wherein the signal peptide is the human kappa light chain signal peptide L1.

3. The T cells according to claim 1 wherein the bispecific antibody is characterized in that: the anti-CD19 light chain variable domain V.sub.LCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 3, the anti-CD19 heavy chain variable domain V.sub.HCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 5, the anti-CD3 heavy chain variable domain V.sub.HCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 7 and the anti-CD3 light chain variable domain V.sub.LCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 9.

4. The T cells according to claim 1 wherein the bispecific antibody further comprises a linker.

5. The T cells according to claim 1 wherein the bispecific antibody is at least 95% identical to the amino acid sequence SEQ ID NO: 1.

6. An ex vivo method of preparation of T cells secreting a bispecific antibody, the method comprising transducing isolated T cells from a subject with a synthetic polynucleotide encoding a bispecific antibody connected to a signal sequence, or a vector comprising the same, wherein the bispecific antibody comprises: an anti-CD19 single-chain fragment variable (scFv) comprising an anti-CD19 light chain variable domain V.sub.LCD19 and an anti-CD19 heavy chain variable domain V.sub.HCD19, an anti-CD3 single-chain fragment variable (scFv) comprising an anti-CD3 heavy chain domain V.sub.HCD3 and an anti-CD3 light chain domain V.sub.LCD3, wherein the domains are arranged in the order V.sub.LCD19-V.sub.HCD19-V.sub.HCD3-V.sub.LCD3, wherein said bispecific antibody comprises: an anti-CD19 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 10, an anti-CD19 light chain CDR2 region consisting of the amino acid sequence Ile-Ala-Ser, an anti-CD19 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 11, an anti-CD19 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 12, an anti-CD19 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 13, an anti-CD19 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 14, an anti-CD3 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 15, an anti-CD3 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 16 and an anti-CD3 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 17, an anti-CD3 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 18, an anti-CD3 light chain CDR2 region consisting of the amino acid sequence Asp-Thr-Ser, an anti-CD3 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 19, and wherein the signal peptide is selected from a human kappa light chain signal peptide or a human lambda light chain signal peptide.

7. The method according to claim 6 wherein the signal peptide of the bispecific antibody is the human kappa light chain signal peptide L1.

8. The method according to claim 6 wherein the bispecific antibody is characterized in that: the anti-CD19 light chain variable domain V.sub.LCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 3, the anti-CD19 heavy chain variable domain V.sub.HCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 5, the anti-CD3 heavy chain variable domain V.sub.HCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 7 and the anti-CD3 light chain variable domain V.sub.LCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 9.

9. The method according to claim 6 wherein the bispecific antibody further comprises a linker.

10. The method according to claim 6 wherein the bispecific antibody is at least 95% identical to the amino acid sequence SEQ ID NO: 1.

11. T cells obtainable according to the method of claim 6.

12. A method for the treatment of a hematological malignancy or an autoimmune disorder in a subject, the method comprising administering to the subject T-cells characterized by secreting a bispecific antibody comprising: an anti-CD19 single-chain fragment variable (scFv) comprising an anti-CD19 light chain variable domain V.sub.LCD19 and an anti-CD19 heavy chain variable domain V.sub.HCD19, an anti-CD3 single-chain fragment variable (scFv) comprising an anti-CD3 heavy chain domain V.sub.HCD3 and an anti-CD3 light chain domain V.sub.LCD3, wherein the domains are arranged in the order V.sub.LCD19-V.sub.HCD19-V.sub.HCD3-V.sub.LCD3, wherein said bispecific antibody comprises: an anti-CD19 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 10, an anti-CD19 light chain CDR2 region consisting of the amino acid sequence Ile-Ala-Ser, an anti-CD19 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 11, an anti-CD19 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 12, an anti-CD19 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 13, an anti-CD19 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 14, an anti-CD3 heavy chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 15, an anti-CD3 heavy chain CDR2 region consisting of the amino acid sequence SEQ ID NO: 16 and an anti-CD3 heavy chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 17, an anti-CD3 light chain CDR1 region consisting of the amino acid sequence SEQ ID NO: 18, an anti-CD3 light chain CDR2 region consisting of the amino acid sequence Asp-Thr-Ser, an anti-CD3 light chain CDR3 region consisting of the amino acid sequence SEQ ID NO: 19 wherein the bispecific antibody is expressed in the cells from a polynucleotide encoding the bispecific antibody and a signal peptide selected from a human kappa light chain signal peptide or a human lambda light chain signal peptide.

13. The method according to claim 12 wherein the signal peptide of the bispecific antibody is the human kappa light chain signal peptide L1.

14. The method according to claim 12 wherein the bispecific antibody is characterized in that: the anti-CD19 light chain variable domain V.sub.LCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 3, the anti-CD19 heavy chain variable domain V.sub.HCD19 is at least 95% identical to the amino acid sequence SEQ ID NO: 5, the anti-CD3 heavy chain variable domain V.sub.HCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 7 and the anti-CD3 light chain variable domain V.sub.LCD3 is at least 95% identical to the amino acid sequence SEQ ID NO: 9.

15. The method according to claim 12 wherein the bispecific antibody further comprises a linker.

16. The method according to claim 12 wherein the bispecific antibody is at least 95% identical to the amino acid sequence SEQ ID NO: 1.

17. The method according to claim 12 wherein said hematological malignancy is selected from the group consisting of acute B cell lymphoblastic leukemia (ALL), minimal residual disease-positive ALL, chronic lymphatic leukemia (CLL), hairy cell leukemia, non-Hodgkin's lymphoma, B cell lymphoma, mantle cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, marginal zone B cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, myelodysplastic syndrome (MDS) and multiple myeloma.

18. The method according to claim 12 wherein the method is combined with a CAR-T treatment comprising administering CAR-T cells to said subject.

19. A pharmaceutical composition comprising the T cells according to claim 1 and at least one pharmaceutically acceptable excipient.

20. The method according to claim 12 wherein said autoimmune disorder is systemic lupus erythematosus.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1. Schematic diagrams showing the genetic (A) and domain structure (B) of the A3B1-OKT3 bispecific antibody (also known as A3B1 BiTE), bearing a signal peptide from the human k light chain signal peptide (S), the anti-CD19 A3B1 scFv gene and the anti-CD3 OKT3 scFv gene, and the His tag (T). The left arrow indicates the direction of transcription. (C) Three-dimensional structure of the A3B1-OKT3 bispecific antibody.

(2) FIG. 2. Detection of secreted A3B1-OKT3 bispecific antibody in the conditioned media from GFP- or A3B1-OKT3 BiTE-transfected HEK-293 cells by western blot (A). The functionality of secreted A3B1-OKT3 BiTE was demonstrated by FACS on CD3.sup.+ Jurkat cells, CD19.sup.+ NALM6 and Raji cells, and CD3.sup.CD19.sup. K562 cells (B) and using different amounts of purified blinatumomab as control (C).

(3) FIG. 3. Analysis of CD3 and CD19 expression on different cell lines by flow cytometry.

(4) FIG. 4. Activation of human primary T cells by secreted A3B1-OKT3 bispecific antibody. FACS analysis of CD69 expression (A), and ELISA for IL-2 secretion (B) of effector (E) Jurkat T cells co-cultured with CD19.sup. (K562) or CD19.sup.+ (Raji or Nalm6) target (T) cells at a 4:1 E:T ratio. In panel (B), bars marked as 1 correspond to GFP, bars marked as 2 correspond to A3B1-OKT3 bispecific antibody and bars marked as 3 correspond to blinatumomab.

(5) FIG. 5. Reducing SDS-PAGE of the purified A3B1-OKT3 bispecific antibody and blinatumomab (A). Titration ELISA of purified A3B1-OKT3 bispecific antibody against plastic-immobilized human CD19-Fc chimera (hCD19) and BSA (B). Induction of T cell cytotoxicity by purified A3B1-OKT3 bispecific antibody against luciferase-expressing CD19.sup.+ Nalm6 and CD19- HeLa cells (C).

(6) FIG. 6. Determination of the vector copy number (VCN) of integrated lentiviruses in Jurkat T cells transduced at different MOIs with A3B1-OKT3 CAR-encoding lentiviruses (bars marked as 2) of A3B1-OKT3 BiTE-encoding lentiviruses (bars marked as 1) (A). Analysis of the intracellular levels of endogenous CD3 and CAR in non-transduced (NT) Jurkat cells, STAb-T and CAR-T cells (B). Determination of the intracellular levels of A3B1-OKT3 bispecific antibody in NT, STAb-T and CAR-T cells (C). Detection of secreted A3B1-OKT3 bispecific antibody in the conditioned media from NT, STAb-T and CAR-T cells (D).

(7) FIG. 7. Non-transduced (NT) or lentivirally-transduced CAR and STAb-T cells were stained to detect cell surface-expressed CAR or cell surface-bound A3B1-OKT3 bispecific antibody (A). Adhesion of NT or lenvirally-transduced CAR-T and STAb-T cells to plastic-immobilized BSA or hCD19 (B).

(8) FIG. 8. (A) Detection of secreted A3B1-OKT3 bispecific antibody in the conditioned media from lentivirally-transduced human primary T cells (STAb-T) by western blot. Conditioned media from non-transduced T cells (NT-T) and blinatumomab were used as negative and positive controls, respectively. (B) Non-transduced (NT-T) or lentivirally-transduced CAR-T and STAb-T cells were stained to detect cell surface-expressed CAR or cell surface-bound A3B1-OKT3 bispecific antibody. (C) NT-T, CAR-T or STAb-T cells were co-cultured with luciferase expressing CD19-negative HeLa.sup.Luc or CD19-positive Nalm6.sup.Luc tumor cells at a 2:1 E:T (C, E). As controls, non-activated T cells were cultured with target cells in the presence of 100 ng/ml blinatumomab (D, F). After 48 hours, IFN- production was determined by ELISA (C, D) and D-luciferin was added to detect bioluminescence (E, F). Percent viability was calculated relative to the luminescence from an equal number of target cells co-cultured with NT-T cells, and used to calculate percent specific lysis (E, F). Data are meanSD (n=3). Determination of the cytotoxic activity of NT-T (points and curves marked as 1), CAR-T (points and curves marked as 2) or STAb-T cells (points and curves marked as 3) co-cultured with CD19-negative tumor cells at a decreasing E:T ratio (G). The experiments were performed three times and results of one representative experiment are shown.

(9) FIG. 9. Decreasing numbers of Cell Trace Violet-stained activated T (A-T) cells (NT-T, CAR-T or STAb-T cells) were co-cultured with Nalm6.sup.Luc or HeLa.sup.Luc target cells (A) and increasing numbers of CFSE-stained freshly isolated non-activated T cells (T.sub.h0). The total effector:target (E:T) ratio was constant (2:1), but the ratios A-T:Target and A-T:T.sub.h0 varied as indicated. After 5 days, Cell Trace Violet or CFSE dilution were analyzed by flow cytometry (B) to measure proliferation of A-T cells (peaks without any mark) and T.sub.h0 cells (peaks marked as 1), respectively. Decreasing numbers of NT-T, CAR-T or STAb-T cells were co-cultured (A) with Nalm6.sup.Luc or HeLa.sup.Luc target cells and increasing numbers of T.sub.h0 cells, from the same donor. The total effector:target (E:T) ratio was constant (2:1), but the ratios activated T cells (A-T):T.sub.h0 and A-T:target varied considerably. After 48 hours, IFN- production was determined by ELISA (C) and D-luciferin was added to detect bioluminescence (D). Percent viability was calculated relative to the luminescence from an equal number of target cells co-cultured with NA-T cells and used to calculate percent specific lysis (D). Data are meanSD (n=3). The experiments were performed three times and results of one representative experiment are shown. Similar experiments were performed in a non-contacting co-culture transwell assay (E). 510.sup.4 target cells (Nalm6.sup.Luc or HeLa.sup.Luc) and 110.sup.5 T.sub.h0 cells were plated in the bottom well and decreasing numbers (from 10.sup.5 to 10.sup.1) of A-T (NT-T, CAR-T or STAb-T) cells in the insert well. After 48 hours, IFN- production was determined by ELISA (F) and the number of live target cells determined by luciferase assay (G). In panels (C), (D), (F) and (G), closed circles and curve marked as 1 corresponds to NT-T+Nalm6, closed circles and curve marked as 2 correspond to CAR-T+Nalm6, closed circles and curve marked as 3 correspond to STAb-T+Nalm6, open circle marked as 4 correspond to NT-T+HeLa, open circle marked as 5 correspond to CAR-T+HeLa and open circle marked as 6 correspond to STAb-T+HeLa. (n=3; *P0.05, **P0.01, ***P0.001, Student's t test).

(10) FIG. 10. Jurkat STAb and CAR cell immunological synapse (IS) assembly. CAR-Jurkat (CAR-JK) and STAb-Jurkat (STAb-JK) cells were stimulated for 15 min with Raji cells. As activation controls, non-transduced Jurkat (NT-Jurkat, NT-JK) cells were incubated with Raji cells loaded with SEE or blinatumomab 5 nM (BLI). SEE.sup. control samples correspond to NT-JK cells incubated with unloaded Raji cells. (A) Distribution of CD3 and actin at the mature IS. Representative cell conjugates of Jurkat cells interacting with Raji cells labelled with CMAC are shown. The green (CD3) and red (actin) channels, as well as the merged images, are shown. Scale bar corresponds to 5 m. The IS topology obtained from the 3D reconstructions of region of interest placed at the IS in confocal stacks containing the red and the green channels are shown. (B) The graph represents the actin clearance at the IS in each sample estimated as the fraction of actin cleared area. Symbols in each sample indicate individual cells analyzed and the black line the average value. Samples were compared by an ordinary one-way ANOVA with a Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0,0001. (C) Graph representing the percentage of cell conjugates showing peripheral CD3 microclusters or central supramolecular activation dusters (cSMAC) formation by CD3 coalescence. Contingency tests were performed in each possible comparison. *p<0.05; **p<0.01; ***p<0.001; ****p<0,0001. (B and C) Analysis from 3 independent experiments.

(11) FIG. 11. Early signaling in Jurkat CAR and STAb cells. (A) Western blot for quantification of PLC1 and ERK1/2 activation. (B) Phosphorylated fraction of the molecules analyzed in (A), normalized to the maximum fraction found in 0 minutes (min). MeanSD from 3 independent experiments is shown. Samples were compared by a paired two-tailed Student t-test. *p<0.05.

(12) FIG. 12. (A-F) Immunological synapse assembly by primary CAR-T and STAb-T cells. Human primary CAR-T and STAb-T cells were stimulated for 15 min with Raji cells. (A) Distribution of CD3 and actin at the mature IS. Representative cell conjugates of T cells interacting with Raji cells labelled with CMAC are shown. The green (CD3) and red (actin) channels, as well as the merged images, are shown. Scale bar corresponds to 5 m. The IS topology obtained from the 3D reconstructions of region of interest placed at the IS in confocal stacks containing the red and the green channels are shown. (B) Graph representing the percentage of cell conjugates showing peripheral CD3 microclusters (in black) or cSMAC formation by CD3 coalescence (in white). Graphs representing CD3 polarization (C), actin polarization (D), CD3/actin colocalization (E) and actin clearance at the IS (F). Symbols in each sample indicate individual cells analyzed and the black line the average value. Samples were compared by an ordinary one-way ANOVA with a Tukey's multiple comparison test. *p<0.05; **p<0.01; ***p<0.001; ****p<0,0001. *p<0.05; **p<0.01; ***p<0.001; ****p<0,0001. (B-F) Analysis from 3 independent experiments.

(13) FIG. 13. Impedance-based real-time cytotoxicity assay. (A) CD19 expression by HEK-293 (line with circles) and HEK-293.sup.CD19 (line with squares) target cells and cell viability kinetics over time of both cell lines cultured alone. (B) Cell viability kinetics of HEK-293.sup.CD19 (upper panel) and HEK-293 (lower panel) co-cultured with different E:T ratios (line 1, E:T ratio 5:1; line 2, E:T ratio 1:1, line 3, E:T ratio 0.5:1, line 4, E:T ratio 0.12:1, line 5, E:T ratio 0.25:1) of NT-T, CAR-T or STAb-T cells, presented as percentage lysis as well (E:T ratio=0.5:1). (C). Cell viability of CAR-T (line 1) and STAb-T (line 2) cells was evaluated over 72 h with measurements taken at 15 min intervals. Cell Index values were determined and normalized. Results from one of two experiments performed in duplicate are shown.

(14) FIG. 14. Leukemia escape from immune pressure. NT-T, CAR-T or STAb-T cells (CD3+CD19) were co-cultured with Nalm6 (CD3CD19.sup.+) target cells at the indicated effector:target (E:T) ratios. Expression of CD19 and CD3 was analyzed, by flow cytometry, in cell co-cultures at time 0 and after 3, 6, 9 and 22 days. (A) Percentage of: T cells (CD3+CD19), wild type leukemic cells (CD3CD19.sup.+) and leukemic cells that have lost CD19 expression (CD3CD19) at the indicated time points. Data are mean of 3 similar experiments. (B) Representative dot plots showing the different cells populations present, after the indicated times, in CAR-T/Nalm6 cell co-cultures performed at an E:T ratio 1:16.

(15) FIG. 15. Modulation of cell surface by transduced Jurkat T cells. Jurkat T cells were transduced (MOI 5) with an A3B1-CAR or A3B1-OKT3 bispecific antibody encoding a lentiviral vector, to obtain CAR-JK or STAb-JK cells, respectively. (A) Level of CAR expression was detected with anti-mouse-Fab mAb and BiTE cell-surface decoration was detected with an anti-His mAb. (B) Non-transduced Jurkat T cells (NT-JK), CAR-JK or STAb-JK were co-cultured with Nalm6 cells at 1:1 E:T ratio and surface expression of CD3, CD19, CD2 and CD10 were analyzed at time 0 and after 2 h. (C) CD19 cell-surface and intracellular expression on Nalm6 cells co-cultured for 2 hours with STAb-JK cells in the absence or the presence of bafilomycin A1.

DESCRIPTION OF EMBODIMENTS

(16) Material and Methods

(17) Cell Lines and Culture Conditions

(18) HEK293 (CRL-1573), HEK293T (CRL-3216), and HeLa (CCL-2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Walkersville, MD, USA) supplemented with 2 mM L-glutamine (Life Technologies, Paisley, UK), 10% (vol/vol) heat inactivated fetal calf serum (FCS) and antibiotics (100 units/mL penicillin, 100 g/mL streptomycin) (both from Sigma-Aldrich, St. Louis, MO, USA), referred as to DMEM complete medium (DCM). Jurkat, Clone E6-1 (TIB-152), Raji (CCL-86), NALM6 (CRL3273) and K562 (CCL-243) cells were maintained in RPMI-1640 (Lonza) supplemented with 2 mM L-glutamine, heat-inactivated 10% FCS, and antibiotics, referred as to RPMI complete medium (RCM). All cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA), and were grown at 37 C. and 5% CO.sub.2. NALM6 cells expressing firefly luciferase (Luc) (NALM6.sup.Luc) were provided by Dr. Manel Juan (Department of Immunology, Hospital Clinic, Barcelona, Spain). HeLa.sup.Luc are HeLa cells infected with lentivirus encoding the Luc gene. All cell lines were routinely screened for mycoplasma contamination by PCR using the Mycoplasma Gel Detection Kit (Biotools, Madrid, Spain).

(19) Jurkat cells were derived from the peripheral blood of a subject with T cell leukemia. Nalm6 cells were derived from the peripheral blood of a subject with acute lymphoblastic leukemia (ALL). Raji cells were derived from the B-lymphocytes of a subject with Burkitt's lymphoma.

(20) Vector Construction and Preparation of Lentiviral Particles

(21) To construct the expression vector pCDNA3.1-A3B1-OKT3 a synthetic gene (A3B1-OKT3 BiTE) encoding the human kappa light chain signal peptide L1, the A3B1 scFv (V.sub.L- V.sub.H), a five-residue linker (G.sub.4S), the OKT3 scFv (V.sub.H- V.sub.L), and a C-terminal polyHis (HHHHHH) tag was synthesized by GeneArt AG (ThermoFisher Scientific, Regensburg, Germany), and cloned as HindIII/XbaI into the plasmid pCDNA3.1 (ThermoFisher Scientific). To generate the lentiviral transfer vector the complete A3B1-OKT3 bispecific antibody gene (including signal peptide L1, A3B1 scFv, (G.sub.4S), OKT3 scFv and polyHis tag) was synthesized by GeneArt AG and cloned as MluI/BspEI into the vector pCCL-EF1-CAR19, to obtain the plasmid pCCL-EF1-A3B1 BiTE. To produce lentiviral particles, HEK293T cells were transfected with the transfer vector together with packaging plasmids pMDLg/pRRE (Addgene, 12251), pRSVrev (Addgene, 12253), and envelope plasmid pMD2.G (Addgene, 12259), using linear polyethyleneimine (PEI) molecular weight (MW) 25,000 (Polysciences, 23966-1).

(22) HEK293T cells (610.sup.6) were plated 24 hours before transfection in 10-cm dishes. At the time of transfection, 14 g total DNA (6.9 g transfer vector, 3.41 g pMDLg/pRRE, 1.7 g pRSV-Rev, and 2 g pMD2.G) was diluted in serum-free DMEM. 35 g PEI was added to the mix and incubated for 20 min at room temperature. After incubation, DNA-PEI complexes were added onto the cells cultured in 7 mL complete DMEM. Media were replaced 4 hours later. Viral supernatants were collected 48 hours later and clarified by centrifugation and filtration using a 0.45-m filter. Viral supernatants were concentrated using ultracentrifugation at 26,000 rpm for 2 hours 30 min. Virus-containing pellets were resuspended in complete XVivo15 media and stored at 80 C. until use.

(23) Viral supernatants were collected 48 hours later and clarified by centrifugation and filtration using a 0.45 m-pore filter, ultracentrifuged (26,000 rpm for 2 hours), resuspended in complete XVivo15 media (Lonza, Walkersville, MD, USA), aliquoted and stored at 80 C. until use.

(24) Expression and Purification of the A3B1-OKT3 Bispecific Antibody

(25) HEK293 cells were transfected with the expression vector using calcium phosphate and selected in DCM with 500 g/ml G-418 (Life Technologies, Grand Island, NY, USA) to generate stable cell lines. Supernatants from transiently and stably transfected cell populations were analyzed by western blotting and FACS. The A3B1-OKT3 bispecific antibody was purified from DCM conditioned medium collected from stably transfected HEK293 cells. Collected medium was centrifuged, filtered and purified 500 mL at a time using a 1 mL HiTrap Excel column (GE Healthcare, Uppsala, Sweden), washing with 30 column volume of 20 mM imidazole in PBS (pH 7.4) and eluting with 500 mM of imidazole in PBS. Elution fractions containing the relevant proteins were then pooled, dialyzed against PBS, and concentrated using spintrap columns (Merck Millipore, Billerica, MA, USA).

(26) Western Blotting

(27) Samples were separated under reducing conditions on 10-20% Tris-glycine gels (Life Technologies, Carlsbad, CA, USA) and transferred onto PVDF membranes (Merck Millipore, Tullagreen, Carrigtwohill, Ireland) and probed with anti-His mAb (Quiagen, Hilden Germany) (200 ng/ml), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse (GAM) IgG, Fc specific (1:5000 dilution) (Sigma-Aldrich). Visualization of protein bands was performed with Pierce ECL Western Blotting substrate (Rockford, IL, USA).

(28) For analysis of BiTE and CAR expression, samples were lysed for 5 minutes in ice-cold RIPA buffer (Sigma-Aldrich) with 5 mM EDTA and 1 Halt Protease Inhibitor Cocktail (ThermoFisher). Lysates were then centrifuged at 11,000 g for 10 minutes at 4 C. and soluble fractions were collected. For analysis of BiTE secretion by transfected or transduced cells, supernatants were collected. 15 mg of protein or 16 ml of supernatant were separated under reducing conditions on 10-20% Tris-glycine gels (Life Technologies, Carlsbad, CA, USA), transferred onto Immobilon-PVDF membranes (Merck Millipore, Tullagreen, Carrigtwohill, Ireland) and probed with mouse anti-human CD247 (1:1000) (BD Biosciences) or anti-His mAb (Qiagen, Hilden Germany) (200 ng/ml), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse (GAM) IgG, Fc specific (1:5000) (Sigma-Aldrich). Mouse anti-b actin HRP-conjugated mAb (1:50,000) was used as loading control. Visualization of protein bands was performed with Pierce ECL Western Blotting substrate (Rockford, IL, USA).

(29) For analysis of T cell signaling, CAR-JK and STAb-JK cells were incubated at 37 C. with Raji cells at a Jurkat/Raji ratio 10:1 for the indicated times. NT-JK cells were incubated with non-loaded Raji cells or Raji cells loaded during 1 hour with 5 nM Blincyto (blinatumomab) (Amgen Inc, Thousand Oaks, California) or 1 g/mL Staphylococcus Aureus Enterotoxin-E (SEE) (Toxin Technologies, Sarasota, FL, USA). The stimulation time 0 minutes corresponds to Jurkat cells mixed with Raji cells at room temperature and immediately centrifuged and lysed. Samples were lysed for 30 minutes in ice-cold lysis buffer containing 20 mM Tris-HCl pH 7.5; 1% NP-40; 0.2% Triton X-100 (Sigma-Aldrich); 2 mM EDTA; 150 mM NaCl; 1.5 mM MgCl.sub.2; 5 mM -glicerolphosphate; lx protease inhibitor cocktail; 1 mM NaF; 1 mM PMSF; 1 mM Na.sub.3VO.sub.4 and 1 mM sodium pyrophosphate. Lysates were then centrifuged at 10.000 rpm for 10 minutes at 4 C. and soluble fractions were collected, mixed with 6 Laemmli buffer (Alfa Aesar, Haverhill, MA, USA) containing 20% -mercaptoethanol, boiled at 95 C. for 5 minutes and resolved in 10% SDS-PAGE acrylamide gels. Resolved proteins were transferred to Immobilion PVDF membranes, which were blocked with blocking buffer (LI-COR Bioscience, Lincoln, NE, USA), incubated over night with rabbit anti phospho-Y783-PLC1, anti PLC1, anti phospho-T202/T204-ERK1/2 or mouse anti-ERK1/2 primary antibodies (all from Cell Signaling Technology, Beverly, MA, USA) and incubated 30 minutes with 680 goat-anti rabbit and 800 goat-anti mouse IR dyes (Miltenyi Biotec). All blots were scanned, and fluorescence was quantified with an Odyssey Infrared Imager (LI-COR). Densitometry of images was done with Image Studio Freeware (LI-COR). When necessary, blots were striped in 50 ml containing 2% SDS; 12.5% Tris-HCl pH 6.8 and 0.7% -mercaptoethanol for 30 minutes at 50 C.

(30) Enzyme-Linked Immunosorbent Assay

(31) Human CD19:human Fc chimera (CD19:Fc) (R&D Systems, Minneapolis, MN, USA) was immobilized (5 g/ml) on Maxisorp plates (NUNC, Roskilde, Denmark) overnight at 4 C. After washing and blocking, conditioned media or purified protein solution were added and incubated for 1 hour at room temperature. The wells were washed and anti-His mAb (Qiagen) added (1 g/ml). After washing, HRP-GAM IgG, Fc specific (1:2000 dilution) (Sigma-Aldrich) was added, and the plate was developed using tetramethylbenzidine (TMB) (Sigma-Aldrich).

(32) Flow Cytometry

(33) The following mAbs against human proteins, were used: FITC-conjugated anti-CD3 (clone UCHT1), APC-conjugated anti-CD3 (clone UCHT1), PE/Cy7-conjugated anti-CD8 (clone RPA-T8), PE-conjugated anti-CD69 (clone L78), all from BD Biosciences (San Jose, CA, USA); PC7-conjugated anti-CD19 (clone J4.119, Beckman Coulter, Marseille Cedex, France); and PE-conjugated anti-CD4 (clone MEM-241, Immunotools, Friesoythe, Germany). Dapi (Sigma-Aldrich) was used as viability marker. Cell surface expression of A3B1 CAR was analyzed using an APC-conjugated F(ab).sub.2 goat anti-mouse IgG F(ab).sub.2 (Jackson ImmunoResearch, West Grove, PA, USA). Cell surface-bound A3B1-OKT3 bispecific antibody was detected with 1 g/ml anti-6His tag-biotin mAb (clone HIS.H8, Invitrogen, Rockford, IL, USA), and PE-conjugated streptavidin (BD Biosciences). Cell acquisition was performed in a BD FACSCAnto II flow cytometer using BD FACSDiva software (both from BD Biosciences, San Jose, CA, USA). Analysis was performed using FlowJo V10 software (Tree Star, Ashland, OR, USA).

(34) Lentivirus Titration

(35) All lentivirus stocks were normalized for p24 and RNA. The p24 concentration was determined by ELISA (Takara, Saint-Germain-en-Laye, France), and the genomic lentiviral RNA by qRT-PCR (Takara). In the case of A3B1 CAR-encoding lentivirus, functional titers (TU/ml) were determined by FACS analysis after limiting dilution in HEK293T cells, using an APC-conjugated F(ab).sub.2 fragment goat anti-mouse IgG F(ab).sub.2 fragment specific (Jackson ImmunoResearch Laboratories).

(36) T Cell Transduction and Culture Conditions

(37) Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of volunteer healthy donors by density gradient centrifugation using Lymphoprep solution (Axis-Shield, Oslo, Norway). All donors provided written informed consent in accordance with the Declaration of Helsinki. CD3+ T cells were purified by negative selection using the Pan T Cell Isolation Kit, human and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany) following the manufacturer's instructions. The purity of isolated populations was routinely >95%. Cells were then activated and expanded for 24 hours using anti-CD3/CD28 beads (Dynabeads, Gibco) at 1:3 cell:bead ratio in RCM, at a concentration of 110.sup.6 cells/ml. 24 hours later cells were left untransduced (NT cells) or transduced with A3B1 CAR (CAR-T cells) or A3B1-OKT3 bispecific antibody (STAb-T cells) encoding lentivirus at the indicated MOI (Multiplicity of Infection, ratio of lentiviral particles:targets). A3B1 CAR-T cells were transduced with a lentiviral vector encoding an anti-CD19 second generation 4-1BB/CD3 CAR (Castella et al, 2019). A period of cell expansion of 6-8 days was carried out before conducting experiments. Three different cell transductions using three different PBMC donors were used to conduct the experiments in triplicate.

(38) T Cell Activation Assays

(39) For Jurkat T cell activation assays, 10.sup.5 Jurkat cells were co-cultured with Raji, Nalm6 or K562 cells at effector:target (E:T) ratio of 2:1, and 50 l of supernatant from transfected HEK293 cell cultures was added. After 48 hours, CD69 expression was analyzed by flow cytometry, and culture supernatants were collected and analyzed for IL-2 levels by ELISA (Diaclone, Besanon Cedex, France.) For primary T cell activation assays, anti-CD3/CD28 beads were removed five days after lentiviral transduction, and activated T cells (A-T) were left resting for 24 hours at a concentration of 0.810.sup.6 cells/ml. Then, transduced or non-transduced A-T were co-cultured with freshly isolated T cells (non-activated T cells, T.sub.h0) from the same donor, and Nalm6 or HeLa target cells at the indicated ratios. As controls, T.sub.h0 cells were cultured with Nalm6 or Hela target cells in the absence or the presence of 100 ng/ml blinatumomab. After 48 hours, supernatants were collected and levels of IFN- were analyzed by ELISA (Diaclone). For proliferation assays, de-beaded A-T were stained with 2.5 M Cell Trace Violet, and freshly isolated T cells were stained with 2.5 M Cell Trace CFSE (both from Life Technologies, Eugene, OR, USA) following manufacturer's instructions. Stained A-T and T.sub.h0 cells were co-cultured with Nalm6 or HeLa target cells at the indicated ratios. After 5 days, samples were acquired in a FACSCanto flow cytometer and T cell proliferation was analyzed using FCS Express 6 Plus Software (De Novo Software, Los Angeles, CA, USA).

(40) Cytotoxicity Assays

(41) As indicated above, five days after lentiviral transduction, anti-CD3/CD28 beads were removed and A-T cells were left resting for 24 hours. Then, transduced or non-transduced A-T cells were co-cultured with freshly isolated T.sub.h0 cells and NALM6.sup.Luc or HeLa.sup.Luc target cells at the indicated ratios. As controls, T.sub.h0 cells were cultured with Nalm6 or Hela target cells in the absence or the presence of 100 ng/ml blinatumomab. After 48 hours viability was measured adding D-luciferin (Promega, Madison, WI, USA) to a final concentration of 20 g/ml and bioluminescence quantified in relative light units using a Victor luminometer (PerkinElmer, Waltham, MA, USA). A 100% lysis control was included by treating the target cells with 1% Triton-X100 (Sigma-Aldrich), and the value for spontaneous lysis was obtained by incubating the target cells with effector cells (NA-T) only. Percent tumor cell viability was calculated as the mean bioluminescence of each sample divided by the mean bioluminescence of the input number of control target cells times 100. Specific lysis is the difference in tumor cell viability relative to control (0%).

(42) For cytotoxic studies in transwell assays, polycarbonate filter inserts (4.26 mm diameter) with 0.4 m pores (Corning, Kennebunk, Me, USA) were used. Nalm6.sup.Luc and HeLa.sup.Luc/MKN45.sup.Luc cells (510.sup.4) were plated on bottom wells of 96-well plate with 2:1 E:T ratio (10.sup.5 de-beaded A-T cells: NT, CAR-T or STAb-T cells at the indicated ratios were added to insert wells). After 48 hours bioluminescence quantified as described above and cell culture supernatants were collected for IFN- secretion analysis.

(43) For realtime cytotoxicity assays, wild type HEK293 (HEK WT) or HEK293 cells stably-transfected to express membrane-bound CD19 (HEK-CD19) were plated at a density of 10.sup.4 cells per well in a volume of 50 L in an E-Plate 16 (Acea Biosciences, San Diego, CA). After plating, cells were cultured for 20 h in an xCELLigence RTCA DP system (Acea BioSciences), inside a cell culture incubator at 37 C. and 5% CO.sub.2, with measurements taken at 15 min intervals to measure Cell Index, a measure of electrical impedance across well electrodes resulting from cell adherence. After 20 h, NT-T, STAbT or CAR-T cells, at different E:T ratios, were added in a volume of 100 L per well, with 2 replicate wells per condition. Plates were returned to the xCELLigence and the cytotoxicity was evaluated over 72 h with measurements taken at 15 min intervals. Cell Index values were determined and normalized using RTCA Software 2.0 (Acea Biosciences). Percentage specific lysis was calculated using the following equation: Percentage=[(cell index of UTDcell index of CAR-T cells)/(cell index of UTD)]100.

(44) Immunofluorescence and Confocal Microscopy

(45) NT, CAR and STAb, Jurkat or primary, T cells were incubated for 15 minutes on Poly-L-lysine (Sigma-Adrich)-coated coverslips at 37 C. with Raji or Nalm6 target cells at a T cell/target ratio 1:1. Where indicated, target cells were loaded for 1 hour with 5 nM blinatumomab or 1 g/mL SEE as in Western blot experiments. In order to properly find cell conjugates, target cells were pre-incubated with the fluorescent tracker chloromethyl derivative of aminocoumarin (CMAC) 1 M (Molecular Probes, Eugene, OR, USA). Jurkat/Raji cell conjugates (200.000 cells each) were fixed with 4% paraformaldehyde in PBS for 5 minutes at RT, permeabilized with 0.1% Triton X-100 for 5 minutes at RT and blocked with 10 g/ml human gamma globulin for 20 minutes at RT. Samples were stained with mouse anti-CD3 (T3b clon; kindly provided by Dr. Francisco Sanchez-Madrid, (Hospital Universitario de la Princesa, Madrid, Spain) or with Phalloidin-647 (Molecular Probes) (for filamentous actin detection) for 1 hour at RT. Cells were then washed with TBS and incubated with goat anti-mouse-Ig Alexa 488 (Molecular Probes) secondary antibody at RT for 30 minutes. Finally, coverslips were washed with TBS and distilled water before being mounted with Mowiol (Sigma-Aldrich) medium.

(46) Confocal sections of fixed samples were acquired using an SP-8 laser scanning laser confocal microscopy (Leica Microsystems, Wetzlar, Germany), with a 60/1.35 oil immersion objective. CMAC, A488 and phalloidin-647 were excited by 405, 488 and 633 nm laser lines, respectively. For 3D reconstructions, z-stacks through the complete IS were acquired every 0.3 m. Actin clearance was estimated by the ratio of the area of the central region of the IS depleted of actin versus the complete area of the IS including the actin ring in 3D images. Assessment of CD3 coalescence and central supramolecular activation dusters (cSMAC) formation was assessed by visual inspection of the 3D images. 3D reconstruction and image quantitation were performed with ImageJ freeware (National Institutes of Health, Rockville, MD, USA).

(47) Statistical Analysis

(48) Results of experiments are expressed as meanstandard deviation (SD). Statistical tests were done with Prism 6 (GraphPad Software, USA).

Example 1. Generation and Characterization of A3B1-OKT3 Bispecific Antibody

(49) An anti-CD19/anti-CD3 BiTE antibody, identified herein as A3B1-OKT3 bispecific antibody or as A3B1-OKT3 BiTE, was generated. A3B1-OKT3 bispecific antibody comprises the anti-CD19 A3B1 scFv linked to the anti-CD3 OKT3 scFv in a tandem structure (FIG. 1A, 1B, 1C).

(50) The anti-CD19 A3B1 scFv and the anti-CD3 scFv are separated by a linker (linker 2). The anti-CD19 A3B1 scFv comprises an anti-CD19 light chain variable domain V.sub.LCD19 and an anti-CD19 heavy chain variable domain V.sub.HCD19. The anti-CD3 scFv comprises an anti-CD3 heavy chain variable domain V.sub.HCD3 and an anti-CD3 light chain variable domain V.sub.HCD3. The domains are arranged in the order V.sub.LCD19-V.sub.HCD19-V.sub.HCD3-V.sub.LCD3.

(51) The domains V.sub.LCD19-V.sub.HCD19 are separated by a linker (linker 1) and the domains V.sub.HCD3-V.sub.LCD3 are also separated by a linker (linker 3).

(52) A3B1-OKT3 bispecific antibody further comprises the human kappa light chain signal peptide L1 and a polyhistidine tag. Table 1 provides details of A3B1-OKT3 bispecific antibody sequence features. The 3 CDRs regions of each light and heavy variable domains are identified in Table 1.

(53) TABLE-US-00001 TABLE 1 Sequence A3B1-OKT3 bispecific antibody SEQ ID NO: 1 Human kappa light chain signal SEQ ID NO: 2 peptide L1 A3B1 light chain variable domain SEQ ID NO: 3 Linker 1 SEQ ID NO: 4 A3B1 heavy chain variable domain SEQ ID NO: 5 Linker 2 SEQ ID NO: 6 OKT3 heavy chain variable domain SEQ ID NO: 7 Linker 3 SEQ ID NO: 8 OKT3 light chain variable domain SEQ ID NO: 9 A3B1 light chain CDR1 region SEQ ID NO: 10 A3B1 light chain CDR2 region Ile-Ala-Ser A3B1 light chain CDR3 region SEQ ID NO: 11 A3B1 heavy chain CDR1 region SEQ ID NO: 12 A3B1 heavy chain CDR2 region SEQ ID NO: 13 A3B1 heavy chain CDR3 region SEQ ID NO: 14 OKT3 heavy chain CDR1 region SEQ ID NO: 15 OKT3 heavy chain CDR2 region SEQ ID NO: 16 OKT3 heavy chain CDR3 region SEQ ID NO: 17 OKT3 light chain CDR1 region SEQ ID NO: 18 OKT3 light chain CDR2 region Asp-Thr-Ser OKT3 light chain CDR3 region SEQ ID NO: 19 Artificial gene encoding for SEQ ID NO: 20 A3B1-OKT3 bispecific antibody

(54) The A3B1-OKT3 bispecific antibody was secreted by transfected HEK293 cells, and the migration pattern was consistent with the molecular weight calculated from its amino acid sequence (54.2 kDa without the signal sequence; FIG. 2A). In FACS analysis, CD3+ Jurkat cells, CD19.sup.+ Nalm6 and Raji cells were used.

(55) FACS analysis demonstrated that A3B1-OKT3 BiTE bound to CD3+ Jurkat cells and to CD19.sup.+ Nalm6 and Raji cells, but not to CD3-CD19- K562 cells (FIGS. 2 and 3). When T cells were co-cultured with CD19.sup.+ target cells in the presence of conditioned medium from A3B1-OKT3 BiTE-transfected HEK293 the levels of CD69 expression (FIG. 4A) and IL-2 secretion (FIG. 4B) were similar to those observed when cells were co-cultured with 100 ng/ml blinatumomab.

(56) The A3B1-OKT3 bispecific antibody was purified from conditioned medium from stably transfected HEK293 cells by immobilized metal affinity chromatography, which yields protein (1 mg/L) that was >90% pure, as assessed by SDS/PAGE (FIG. 5A). Antibody titration ELISA analysis showed a specific and dose-dependent binding of A3B1-OKT3 bispecific antibody to plastic immobilized human CD19 in fusion with human Fc (hCD19) (FIG. 5B). The A3B1-OKT3 bispecific antibody could redirect primary T cells to specifically lyse CD19.sup.+ Nalm6 cells in a concentration-dependent fashion with an EC.sub.50 values of 90 pg/mL (FIG. 5C). These values were similar to those determined in the same assay for blinatumomab.

Example 2. Preparation of T Cells Secreting A3B1-OKT3 Bispecific Antibody

(57) The A3B1-OKT3 bispecific antibody was cloned into a lentiviral vector and Jurkat T cells transduced at MOI (Multiplicity of Infection, ratio of lentiviral particles:targets) values of 1, 5 and 10, with A3B1-OKT3 bispecific antibody-encoding or A3B1 CAR-encoding lentiviruses and the relationship between the number of vector integrations and transgene expression analyzed. Vector copy number (VCN) was found to be similar in both cases, between 1 and 5 copies for A3B1 CAR-infected Jurkat T cells (CD19 CAR-Jurkat), and 1 and 7 for A3B1-OKT3 bispecific antibody-infected Jurkat T cells (CD19 STAb-Jurkat) (FIG. 6A). The intracellular levels of both proteins were similar as determined by Western blotting, with a clear correlationship between MOI and i) CAR expression (FIG. 6B) and ii) bispecific antibody expression and secretion (FIG. 6C,D). The A3B1-OKT3 bispecific antibody was detected in conditioned medium from CD19 STAb-Jurkat cells (FIG. 6D). No A3B1-OKT3 bispecific antibody molecules were detected in media from non-transduced (NT) Jurkat cells. The percentage of A3B1 CAR+ Jurkat T cells varied between 65% and 100%, and a VON-dependent surface staining of CD19 STAb-Jurkat cells was observed with an anti-His-tag mAb, ranging from 16 to 78%, indicating that secreted A3B1-OKT3 BiTEs were loaded onto the CD3 complexes on the T cell surface (FIG. 7A). Importantly, the process of cis-/trans-decoration of the CD3 complex by the secreted A3B1-OKT3 BiTE antibodies results in an effective and specific adhesion of STAb-Jurkat cells to immobilized hCD19 (FIG. 7B).

(58) Next, CD3/CD28-activated primary human T cells were transduced to generate CD19 STAb-T cells. Activated T cells were transduced at a MOI (Multiplicity of Infection, ratio of lentiviral particles:targets) of 10 with A3B1-OKT3 bispecific antibody-encoding or A3B1 CAR-encoding lentiviruses, and after an expansion period, the expression of both A3B1-based molecules analyzed. The A3B1-OKT3 bispecific antibody was detected in conditioned-medium from STAb-T cells, had the expected molecular weight (FIG. 8A), and specifically recognize CD19.sup.+ and CD3+ cell lines (not shown). No A3B1-OKT3 bispecific antibodies were detected in media from non-transduced (NT-T) (FIG. 8A) or CAR-T cells. Positive surface staining of STAb-T cells was observed with an anti-His-tag antibody (mean of 45%), indicating that secreted A3B1-OKT3 bispecific antibody are loaded onto the CD3 complexes on the T cell surface (FIG. 8B). The percentage of CAR+ T cells varied between 30% and 76%, depending on the experiment (FIG. 8B).

Example 3. Killing Activity of T Cells Secreting A3B1-OKT3 Bispecific Antibody and CD19 CAR-T Cells

(59) T cells secreting A3B1-OKT3 bispecific antibody (STAb-T cells) secreted IFN after a co-culture at a 2:1 E:T ratio with CD19.sup.+ Nalm6 cells, but not with CD19- HeLa cells (FIG. 8C). These levels were similar to those observed with CAR-T cells (FIG. 80) or when non-transduced (NT) activated T cells (NT-T) were co-cultured with Nalm6 cells in the presence of blinatumomab (FIG. 8D). In cytotoxicity assays, STAb-T cells killed CD19.sup.+ but not CD19-cells (FIG. 8E). NT-T cells had no cytolytic activity confirming the specificity of STAb-T cells. These results indicate that STAb-T cells recognize and kill CD19-positive target cells in antigen dependent manner (FIG. 8E). Furthermore, STAb-T cells were more efficient than CAR-T cells at lower E:T ratios concerning the cytotoxic potential in relation to E:T ratio and time (FIG. 8G).

Example 4. Effect of T Cells Secreting A3B1-OKT3 Bispecific Antibody on Other T Cells

(60) To demonstrate that STAb-T cells were able to recruit bystander T cells to kill CD19-positive targets, different in vitro co-culture setups, with and without direct cell-cell contacts were used (FIG. 9). To this end, NT or lentivirally-transduced activated T cells (A-T) or mixtures of A-T and non-activated T cells (T.sub.h0) were co-cultured with CD19.sup.+ Nalm6 or CD19- HeLa cells at a constant 2:1 E:T ratio. The A-T (NT-T, CAR-T or STAb-T) and T.sub.h0 cells were mixed at different A-T:T.sub.h0 ratios (from 1:9 to 1:100.000) keeping a constant number of 110.sup.5 effector T cells.

(61) When stimulated with Nalm6 cells, in a direct cell-cell contact context (FIG. 9A), the STAb-T cells proliferated efficiently and exhibited a percentage of dividing cells of 76, 86 and 100 at 2:1, 1:1 and 1:50 A-T:Target ratios, respectively (FIG. 9B). The proliferation of CAR-T cells was less efficient at the 2:1 A-T:Target ratio with only a 54% of dividing cells (FIG. 9B). In this context, the levels of IFN- secretion by CAR-T cells were significantly higher than those observed in STAb-T cells (FIG. 90). However, the cytotoxic capacity of CAR-T cells rapidly declined as the A-T:Target ratio increased being able to induce 50% lysis of Nalm6 cells at a 1:50 A-T:Target ratio, which disappeared completely at the 1:500 A-T:Target ratio (FIG. 9D). Importantly, bystander T.sub.h0 cells mixed with STAb-T cells were efficiently stimulated to proliferate by Nalm6 cells, with a percentage of dividing cells of 22.3 and 7.3 at 1:9 and 1:100 A-T:T.sub.h0 ratios, respectively (FIG. 9A).

(62) The percentage of dividing T cells was higher than observed when T.sub.h0 cells were cultured with Nalm6 cells in the presence of blinatumomab. This ability to recruit bystander T.sub.h0 cells results in STAb-T cells being able to induce 100% of tumor cell lysis, even when they represented 1 out of 500 total Target cells (FIG. 9D). Even at a 1:5,000 A-T:Target ratio, STAb-T cells induced 30% lysis of Nalm6 cells (FIG. 9D). No detectable proliferation, IFN- secretion and cytotoxicity was observed when T.sub.h0 cells were mixed with NT, and co-cultured with Nalm6 cells (FIG. 9A-D; respectively); and when A-T cells and A-T:T.sub.h0 mixtures were cultured with HeLa cells (FIG. 9A-D; respectively).

(63) To further demonstrate that STAb-T cells are able to recruit bystander T.sub.h0 cells to CD19-positive targets, transwell assays were used (FIG. 9E). CD19.sup.+ Nalm6.sup.Luc cells or CD19-HeLa.sup.Luc cells were plated with or without T.sub.h0 cells in the bottom well, and NT-T, CAR-T or STAb-T cells were plated in the insert well (FIG. 9E). T cells activation (FIG. 9F) and tumor cell killing (FIG. 9G) was dependent on the presence of T.sub.h0 cells in the bottom well and STAb-T cells in the insert well, indicating that secreted A3B1-OKT3 BiTEs effectively redirected T.sub.h0 cells to CD19-positive targets. The extraordinary recruiting capacity of these cells is evidenced by the fact that only 1000 STAb-T cells were able to induce 100% of tumor cell lysis, indicating that a single STAb-T cell is capable of efficiently redirecting 100 T.sub.h0 cells to kill CD19.sup.+ tumor cells (FIG. 9G).

Example 5. Spatial Topology of the Immunological Synapses Assembled by CAR and STAb Cells

(64) The spatial topology of the immunological synapse (IS) assembled by the A3B1 CAR or the A3B1-OKT3 bispecific antibody was studied in CD19 CAR-Jurkat (CD19 CAR-JK) cells and CD19 STAb-Jurkat (CD19 STAb-JK) cells, using the CD19-expressing cell line Raji as antigen presenting cell. As controls, non-transduced Jurkat (NT-JK) cells were incubated with unloaded (non-activated control) or with Raji cells loaded with blinatumomab or the bacterial superantigen (SAG) staphylococcal enterotoxin E (SEE) (activation controls). Bacterial SAGs bind, as intact molecules to the class II MHC molecules expressed on professional antigen presenting cells (APCs) outside the peptide-binding groove then sequentially bind the TCR via the variable region of the TCR -chain. Jurkat-Raji cell conjugates incubated at 37 C. for 15 minutes were stained for F-actin and CD3 to evaluate the organization of the distal and central supramolecular activation clusters, dSMAC and cSMAC, respectively. 3D confocal microscopy was implemented to visualize the central filamentous actin clearance, with the typical actin ring at the dSMAC, and the coalescence of CD3 microclusters at the cSMAC occurring in the mature IS. All conditions, including CD19 CAR-JK cells, recruited CD3 to the IS. However, STAb-JK, but not CAR-JK cells cleared actin and formed the cSMAC by CD3 coalescence in a similar way to J-NT-T cells stimulated by SEE or blinatumomab (FIG. 10A, 10B, 10C).

Example 6. Early Signaling During CAR- and STAb-Mediated Jurkat T Cell Activation

(65) The activation of CAR-JK and STAb-JK cells was assessed in co-cultures with Raji cells. As activation control, NT-T cells were stimulated with blinatumomab- or SEE-loaded Raji cells. As negative control, NT-JK cells were incubated with Raji cells alone. PLC1 and ERK1/2 activation were analyzed by Western blot due to their important role in early activation signaling downstream the TCR. Interestingly, STAb-JK cells showed PLC1 and ERK1/2 activation kinetics similar to NT-JK cells stimulated with SEE or blinatumomab. However, CAR-JK cells showed a more transient signaling compared to the rest of stimulation conditions (FIG. 11A, 11B).

Example 7. Immunological Synapse Assembly by Primary STAb-T and CAR-T Cells

(66) The immunological synapse assembly by primary STAb-T cells and CAR-T cells following their interaction with Raji cells was analyzed. Primary STAb-T cells organized a canonical IS, with proper filamentous actin (F-actin) containing dSMAC and accumulation of CD3 at the cSMAC (FIG. 12A, 12B, 12C, 12D, 12E, 12F). By contrast, primary CAR-T cells formed a non-canonical IS with disperse clusters of CD3 and a F-actin not properly cleared from the central area of the cognate interaction (FIG. 12A, 12B, 12C, 12D, 12E, 12F).

Example 8. CD19 STAb-T Cells Exhibited Superior Killing Ability than CD19 CAR-T Cells

(67) In an impedance-based cytotoxicity assay for real-time and label-free assessment of T cell-mediated killing primary STAb-T cells were found to mediate rapid reduction in target cell viability against adherent CD19.sup.+ cells (FIG. 13A) at all the effector to target (E:T) ratios tested, whereas primary CAR-T cells showed cytotoxic effect only at the highest E:T ratios (FIG. 13B). When displayed as percentage cytotoxicity at several time points, STAb-T cells were significantly more effective than CAR-T cells (FIG. 13C). By contrast, CD19.sup.+ cells co-cultured with non-transduced (NT)-T cells, or CD19.sup. cells co-cultured with NT-T, CAR-T or STAb-T cells, displayed similar viability kinetics than those showed by target cells cultured alone (FIG. 13B).

Example 9. CD19.SUP.+ Leukemia Cells Escape from Immune Control Exerted by CAR-T and STAb-T Cells

(68) Next, the ability of CD19.sup.+ leukemic B cells to escape from immune control was studied by co-culturing primary CAR and STAb T cells with Nalm6 cells at low E:T ratios. Whereas co-culture with NT-T cells did not inhibit the growth of Nalm6 cells (FIG. 14A), STAb-T cells completely eliminated all leukemia cells from day 6 (FIG. 14A), even at the 1:16 E:T ratio (FIG. 14A). By contrast, CAR-T cells did not eliminate CD19.sup.+ cells at the 1:8 E:T ratio where leukemia cells persist, with a partial down-modulation of CD19 (FIG. 14A). On day 6, Nalm6 cells have almost completely lost surface expression of CD19, a situation that was still evident on day 9. Thereafter, leukemia cells progressed and recover surface expression of CD19 (FIG. 14B).

(69) To more carefully study leukemia escape, Nalm6 cells were co-cultured at 1:1 E:T ratio with lentivirally-transduced Jurkat T cells, CAR-JK or STAb-JK (FIG. 15A). Nalm6 cells showed a rapid and nearly complete CD19 down-modulation after 2 hours co-culture with CAR-JK cells, that did not affect CD10 expression (FIG. 15B). The CAR CD19 molecules were profoundly down-modulated from the Jurkat cell surface after encountering CD19.sup.+ cells, while the expression of CD3 was unaffected (FIG. 15B). CD19 was not lost in co-culture with STAb-JK cells, where a large population of CD3+CD19.sup.+ cells was found, consisting predominantly of CD2.sup.CD10.sup.+ cells (FIG. 15B). Although the CD3 expression level was slightly lower, there were no significant differences in the percentage of CD3.sup.+ cells, but the transfer of CD3 protein from STAb-JK cells to Nalm6 cells was evident (FIG. 15B). To further study the mechanism of CAR-mediated downmodulation CAR-JK cells and Nalm6 cells were co-cultured for two hours either in the absence, or presence of bafilomycin A1 (BafA1), a specific inhibitor of vacuolar H.sup.+-ATPase (V-ATPase). After cell surface CD19 staining with PCy7-labelled anti-CD19 J3-119 mAb, cells were fixed and permeabilized, then stained with PCy5-labelled anti-CD19 J3-119 mAb. As shown in FIG. 15C, BafA1-treated cells expressed significantly higher amounts of intracellular CD19 than untreated cells. These results suggest that CD19 is rapidly internalized and degraded by Nalm6 cells following interaction with the CAR.

(70) Sequence Listing Free Text

(71) The sequence listing contains free text, which is shown in Table 2.

(72) TABLE-US-00002 TABLE 2 SEQ ID NO Position Text 1 A3B1-OKT3 bispecific antibody comprising A3B1 anti-CD19 scFv and OKT3 anti-CD3 scFv 1 . . . 22 Human kappa light chain signal peptide L1 26 . . . 135 A3B1 light chain variable domain 51 . . . 60 A3B1 light chain CDR1 region 78 . . . 80 A3B1 light chain CDR2 region 117 . . . 125 A3B1 light chain CDR3 region 136 . . . 155 Linker 1 157 . . . 279 A3B1 heavy chain variable domain 183 . . . 190 A3B1 heavy chain CDR1 region 208 . . . 215 A3B1 heavy chain CDR2 region 254 . . . 268 A3B1 heavy chain CDR3 region 280 . . . 284 Linker 2 286 . . . 404 OKT3 heavy chain variable domain 311 . . . 318 OKT3 heavy chain CDR1 region 336 . . . 343 OKT3 heavy chain CDR2 region 382 . . . 393 OKT3 heavy chain CDR3 region 405 . . . 525 Linker 3 420 . . . 525 OKT3 light chain variable domain 446 . . . 450 OKT3 light chain CDR1 region 468 . . . 470 OKT3 light chain CDR2 region 507 . . . 515 OKT3 light chain CDR3 region 528 . . . 533 Polyhistidine tag 2 Human kappa light chain signal peptide L1 3 A3B1 light chain variable domain 4 Linker 1 5 A3B1 heavy chain variable domain 6 Linker 2 7 OKT3 heavy chain variable domain 8 Linker 3 9 OKT3 light chain variable domain 10 A3B1 light chain CDR1 region 11 A3B1 light chain CDR3 region 12 A3B1 heavy chain CDR1 region 13 A3B1 heavy chain CDR2 region 14 A3B1 heavy chain CDR3 region 15 OKT3 heavy chain CDR1 region 16 OKT3 heavy chain CDR2 region 17 OKT3 heavy chain CDR3 region 18 OKT3 light chain CDR1 region 19 OKT3 light chain CDR3 region 20 Artificial gene encoding for A3B1-OKT3 bispecific antibody

CITATION LIST

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