REGION

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The Sequence Listing is concurrently submitted herewith with the specification as an ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of November 19, 2019, and a size of 18.7 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

FIELD OF THE INVENTION

The present invention relates to humanized CD 19 ScFv (clone 11) derived from mouse FMC63 antibody, with a mutation in CDR1 region of V _H . The present invention also relates to CD19-CAR-T cells using the humanized CD 19 ScFv of the present invention.

BACKGROUND OF THE INVENTION

Immunotherapy is emerging as a highly promising approach for the treatment of cancer. T cells or T lymphocytes, the armed forces of our immune system, constantly look for foreign antigens and discriminate abnormal (cancer or infected cells) from normal cells. Genetically modifying T cells with CAR (Chimeric antigen receptor) constructs is the most common approach to design tumor-specific T cells. CAR-T cells targeting tumor-associated antigens (TAA) can be infused into patients (called adoptive cell transfer or ACT) representing an efficient immunotherapy approach [1, 2] The advantage of CAR-T technology compared with chemotherapy or antibody is that reprogrammed engineered T cells can proliferate and persist in the patient (“a living drug”) [1, 3, 4]

CARs typically consist of a monoclonal antibody-derived single-chain variable fragment (scFv) at the N-terminal part, hinge, transmembrane domain and a number of intracellular co-activation domains: (i) CD28, (ii) CD 137 (4-1 BB), CD27, or other costimulatory domains, in tandem with an activation CD3-zeta domain. (Figure 1) [1,2]. The evolution of CARs went from first generation (with no co-stimulation domains) to second generation (with one co-stimulation domain) to third generation CAR (with several costimulation domains). Generating CARs with two costimulatory domains (the so-called 3 ^rd generation CAR) have led to increased cytolytic CAR-T cell activity, improved persistence of CAR-T cells leading to its augmented antitumor activity. FIG. 1 illustrates the structures of CAR. The left panel shows the structure of the first generation of CAR (no costimulatory domains). The middle panel shows the structure of the second generation of CAR (one co-stimulation domain CD28 or 4-BB). The right panel shows the third generation of CAR (two or several co-stimulation domains) [6]

Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells, acting at around 3 days after infection, and respond to tumor formation.

CD19

CD 19 is a B-cell type lymphocyte antigen which is expressed on all B-cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and Non-Hodgkin lymphomas. This universal expression among leukemia and lymphomas made this antigen an attractive target for targeting with CAR-T cells [3]

CD19 structure and signaling

The human CD 19 protein is a 95 kDa transmembrane glycoprotein which consists of 556 amino-acids: 20-291 -extracellular domain; 292-313 -transmembrane domain; 314-556- cytoplasmic domain as shown below (extracellular domain underlined). It belongs to immunoglobulin superfamily proteins and mediates B cell receptor, BCR-dependent and independent signaling. CD19 binds to BCR and other cell surface protein to modulate intracellular signaling through binding other kinases and binding partners. CD 19 signaling is connected with Src-family kinase, PI3 Kinase, Abl, AKT-dependent signaling. CD 19 is a biomarker of B-cells mediating survival signaling and immune responses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The structures of CAR

FIG. 2. The structure of humanized CD 19- CAR construct. The humanized CD 19 scFv (clone 11) containing a mutation V27G in CDR1 region of VH is shown. The second generation CD 19-CAR is used.

FIG. 3. The model of humanized CD 19 with a mutation V27G in CDR1 region of

VH.

FIG. 4 Humanized CD 19-CAR construct was detected by FACS analysis with goat anti-human (Fab) ₂ antibody by FACS. Humanized CD 19-C AR-positive cells were detected after transduction of lenti viral humanized CD 19 CAR into T cells.

FIG. 5. Humanized CD19-CAR-T cells killed Hela-CD19 cells but not Hela cells. XCelligence Real-time cytotoxicity assay was used for detection of humanized CD19-CAR-T and mouse CD19-41BB-CD3 CAR-T cell cytotoxicity, as described [4] Normalized cell index is shown on Y-axis, and time in hours is shown on X-axis. From top to bottom on the right, Target cells alone; then effector cells added to target cells: T cells, Mock CAR-T cells, Mouse CD19-CAR-T cells, Humanized CD 19 CAR-T cells.

FIG. 6. Humanized CD19-CAR-T cells secreted IFN-gamma with Hela-CD 19- positive cells. *: p<0.05, IFN-gamma secreted by humanized CAR-T cells versus mouse CD19-CAR-T cells, T cells, Mock CAR-T cells in Hela-CD 19 cells.

FIG. 7. Humanized CD19-CAR-T cells significantly decreased mouse xenograft tumor growth by imaging.

FIG. 8. Humanized CD19-CAR-T cells significantly decreased total flux (imaging signal) of Raji-luciferase xenograft tumors in NSG mice model in vivo. Humanized CAR-T cells and mouse CD19-CAR-T cells showed total flux at about baseline.

FIG. 9. Humanized CD19-CAR-T cells significantly prolonged survival of mice in Raji xenograft model. Kaplan-Meier survival curve shows that humanized (h) CD19-CAR-T cells with mutation (V27G) in CDR1 region of VH significantly prolonged survival of mice versus mouse (m) CD19-CAR-T cells and Mock CAR-T cells. CAR-T cells were injected by i.v 1x10L7 cells/mice. *p= 0.0021, hCD19-41Bb-CD3 CAR-T cells vs Mock CAR-T cells; **: p<0.029, m CD 19-41 BB-CD3 CAR-T cells vs Mock-CAR-T cells.

FIG. 10. Humanized CD 19 CAR-T cells having mutation of V27G in CDRl of V _H prolonged survival of mice comparing with non-mutated humanized CD19 CAR-T cells.

FIG. 11. Humanized CD19-CAR-T cells (clone 11 antibody) and mouse CD19-CAR- T cells (FMC63 antibody) were detected in mouse blood after injecting CAR-T cells into mice. Rabbit anti-FMC63 antibody detected mouse CD19-CAR-T cells; human anti (Fab’a) goat anti -human (Fab)2 antibody detected humanized CD19-CAR-T cells. Control is Mock CAR-T cells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, a "chimeric antigen receptor (CAR)" is a receptor protein that has been engineered to give T cells the new ability to target a specific protein. The receptor is chimeric because they combine both antigen-binding and T-cell activating functions into a single receptor. CAR is a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain, and at least one intracellular domain. The "chimeric antigen receptor (CAR)" is sometimes called a "chimeric receptor", a "T-body", or a "chimeric immune receptor (CIR)." The "extracellul ar domai n capabl e of bi ndi ng to an antigen" means any oligopeptide or polypeptide that can bind to a certain antigen. The "intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell.

“CDR”s are complementary-determining Regions of VH or VL chains of antibody which are critical for binding with antigen.

As used herein,“humanized antibodies” are antibodies from non-human species whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans.

As used herein, a "domain" means one region in a polypeptide which is folded into a particular structure independently of other regions.

As used herein, a "single chain variable fragment (scFv)" means a single chain polypeptide derived from an antibody which retains the ability to bind to an antigen. An example of the scFv includes an antibody polypeptide which is formed by a recombinant DNA technique and in which Fv regions of immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via a spacer sequence. Various methods for engineering an scFv are known to a person skill ed in the art.

As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer.

The inventors have engineered humanized CD 19 scFv starting from heavy and light chain variable regions of mouse monoclonal antibody derived from hybridoma cell line FMC63 [8] The CDRs 1-3 of V _L and CDRs 2 and 3 of V _H of the humanized CD 19 scFv of the present invention are identical to those of the mouse FMC63 antibody. CDR1 of V _H of the humanized CD 19 scFv of the present invention is different from that of the mouse FMC63 antibody, in that it has a mutation of V27G.“ V27G” mutation, as used throughout this application, refers to the mutation at the 27th amino acid residue of V _H , which is the second amino acid residue in CDRl. This mutation was generated by bioinformatics in silico method; the mutation provides a better binding to the CD 19 antigen. By 3D-modeling and binding experiments, the inventors have discovered that the mutation in CDRl region of V _H (V27G) in humanized CD 19 antibody improves the binding of the antibody with CD 19 antigen. Figure 3 depicts a model of humanized CD 19 with a mutation in CDR 1 of V _H (V27G).

The inventors have produced humanized CD19-CAR-T cells to target cancer cells overexpressing CD 19 antigen. The humanized CD19-CAR-T cells of the present invention secrete high level of IFN-gamma against leukemia cancer cells. The humanized CD19-CAR-T cells of the present invention kill Hela-CD 19-positive target cells but do not kill control Hela cells.

The present invention is directed to a humanized anti -human CD 19 antibody derived from mouse clone FMC63, comprising humanized V _H having the amino acid sequence of SEQ ID NO: 2 and humanized V _L having the amino acid sequence of SEQ ID NO: 3, respectively.

In one embodiment, the humanized anti-human CD 19 antibody is a single-chain variable fragment (scFv). ScFv can be V _H -linker- V _L or V _L -linker- V _H .

The present invention is also directed to a chimeric antigen receptor fusion protein comprising from N-terminus to C -terminus: (i) a single-chain variable fragment (scFv) against CD 19 in which V _H has the amino acid sequence of SEQ ID NO 7, and V _L has the amino acid of SEQ ID NO: 6, (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.

In one embodiment, the CAR structure is shown in FIG. 2. In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4- IBB, GITR, ICOS-1, CD27, OX-40 and DAPIO. A preferred the co-stimulatory domain is CD28.

A preferred activating domain is CD3 zeta (CD3 Z or CD3z).

The transmembrane domain may be derived from a natural polypeptide, or may be artificially designed. The transmembrane domain derived from a natural polypeptide can be obtained from any membrane-binding or transmembrane protein. For example, a

transmembrane domain of a T cell receptor a or b chain, a CD3 zeta chain, CD28, CD3e., CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, ICOS, CD 154, or a GITR can be used. The artificially designed transmembrane domain is a polypeptide mainly comprising hydrophobic residues such as leucine and valine. It is preferable that a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide linker or a polypeptide linker, for example, a linker having a length of 2 to 10 amino acids can be arranged between the transmembrane domain and the intracellular domain. In one embodiment, a linker sequence having a glycine-serine continuous sequence can be used.

The present invention provides a nucleic acid encoding the CD 19-CAR. The nucleic acid encoding the CAR can be prepared from an amino acid sequence of the specified CAR by a conventional method. A base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBank for an amino acid sequence of each domain, and the nucleic acid of the present invention can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a nucleic acid can be synthesized, and the nucleic acid of the present invention can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).

A nucleic acid encoding the CAR of the present invention can be inserted into a vector, and the vector can be introduced into a cell. For example, a virus vector such as a retrovirus vector (includi ng an on coretrovirus vector, a lenti virus vector, and a pseudo type vector), an adenovirus vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector or a sendai virus vector, an Epstein-Barr virus (EBV) vector, and a HSV vector can be used. A virus vector lacking the replicating ability so as not to self- replicate in an infected cell is preferably used.

For example, when a retrovirus vector is used, a suitable packaging cell based on a LTR sequence and a packaging signal sequence possessed by the vector can be selected for preparing a retrovirus particle using the packaging cell. Examples of the packaging cell include PG13 (ATCC CRL- 10686), PA317 (ATCC CRL-9078), GP+E-86 and GP+envAm- 12, and Psi-Crip. A retrovirus particle can also be prepared using a 293 cell or a 293T cell having high transfection efficiency. Many kinds of retrovirus vectors produced based on retroviruses and packaging cells that can be used for packaging of the retrovirus vectors are widely commercially available from many companies.

A CAR-T cell binds to a specific antigen via the CAR, thereby a signal is transmitted into the cell, and as a result, the cell is acti vated. The acti vation of the cell expressing the CAR is varied depending on the kind of a host cell and an intracellular domain of the CAR, and can be confirmed based on, for example, rel ease of a cytokine, improvement of a cell proliferation rate, change in a cell surface molecule, or the like as an index. For example, release of a cytotoxic cytokine (a tumor necrosis factor, lymphotoxin, etc.) from the activated cell causes destruction of a target cell expressing an antigen. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

The cell expressing the CAR can be used as a therapeutic agent for a disease. The therapeutic agent comprises the cell expressing the CAR as an active ingredient, and it may further comprise a suitable excipient.

The inventors have generated a humanized anti -human CD 19 antibody, clone 11, and characterized it. Clone 11 exhibits selective and high-affinity binding to human CD 19, and it is used to construct a single-chain variable fragment (scFv). The inventors insert the CD 19 scFv into a second-generation CAR, generate CAR-T cells, and measure their activity against CD19-positive cells in vitro and in a mouse Raji xenograft tumor model. The humanized CD19-CAR-T cells of the present invention secret high levels of IFN-gamma; they are positive by cytotoxicity assay with Hela-CD19 cells but not with Hela cells, which indicates specific killing acti vity of CAR-T cells against target cancer cells with CD19 overexpression. The inventors demonstrate that humanized CD19-CAR-T cells based on clone 11

significantly decreased tumor growth and prolonged survival in mice, which indicates CD19- CAR-T cells can treat patients with CD-19 positive tumors such as B-cell malignancies including leukemia (e.g., acute lymphoblastic leukemia and chronic lymphoblastic leukemia), and lymphomas (e.g., non-Hodgkin lymphomas).

The advantages of the humanized CD19-ScFv (clone 11 antibody) of the present invention vs. mouse CD19-ScFv (FMC63) include less immunogenicity to human due to humanized CD 19 scFv. In addition, the humanized CD 19-CAR T cells of the present invention has better activities in terms of decreasing lymphoma tumor growth and prolonged animal survival than mouse CD 19-CAR T cells.

The humanized CD19-ScFv of the present invention i ncludes a mutati on in CDR1 of V _H (V27G in V _H ) from mouse antibody FMC63. The mutation provides a change in conformation of ScFv in the antigen binding region and results in prolonged survival in mice treated with non-mutated humanized CD 19-CAR T cells.

The present humanized CD 19 ScFv can be used for immunotherapy applications: toxin/drug-conjugated antibody, monoclonal therapeutic antibody, and CAR-T cell immunotherapy.

Humanized CD! 9-CAR-T cells using the present humanized CD19 ScFv effectively target CD 19 antigen in CD 19-positive cancer cell lines.

Humanized CD 19-CAR-T cells can be used in combination with different

chemotherapy: checkpoint inhibitors, targeted therapies, small molecule inhibitors, and antibodies.

Humanized CD 19-CAR-T cells can be used clinically for CD 19-positive cancer cells.

Modifications of co-activation domains such as CD28, 4-1BB and others can be used to increase the efficacy of CAR-T cells. Tag-conjugated humanized CD19 scFv can be used for CAR generation.

Humanized CD 19-CAR-T cells can be used with different safety switches such as t- EGFR, RQR (Rituximab-CD34-Rituximab), inducible caspase-9 and other.

Third generation CAR-T or other co-activation signaling domains can be used for the same humanized CD19-scFv to prepare CD 19-CAR-T.

The humanized CD 19 CAR can be combined with CARs targeting other tumor antigens or tumor microenvironment, e.g., VEGFR-1-3, PDL-1, bi-specific antibodies with CD 19 and CD3, or other antigens can be generated for therapy.

The humanized CD 19-CAR can be used for generating other types of cells such as CAR-natural killer (NK) cells, CD 19-C AR-macrophages, and other CD 19-CAR

hematopoietic cells, which can target CD 19-positive cancers. The present invention provides T cells, or NK cells, or macrophages, or hematopoietic cells, modified to express the CD 19- CAR.

The humanized CD 19-CAR-T cells can be used against cancer stem cells or tumor initiating cells that are most resistant against chemotherapy and form aggressive tumors. The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLES

The inventors generated humanized CD19-ScFv-CAR constructs inside lentiviral vector cloned into Xba I and Eco R I sites of lentiviral vector. pCD510-FMC63-28z lentiviral CAR construct containing the humanized CD 19 ScFv-41BB-CD3zeta insert - between the Xba I and Eco RI cloning sites.

The lentiviruses were generated in 293T cells and titer was established by RT-PCR. Then equal dose of lentiviruses was used for transduction of T cells.

Example 1. Humanized CD19 Antibody: VH and VL and scFv Sequences

The inventors have humanized CD19 scFv from mouse CD19 FMC63 scFv clone [8], and selected a humanized scFv with a mutation in CDR1 (Clone 11) based on the in vivo data efficacy. The structure of humanized CD19 scFv is: VL-linker-VH.

The nucleotide sequence and the amino acid sequence of humanized CD 19 V _L are shown below.

The nucleotide sequence and the amino acid sequence of a linker are shown below.

The nucleotide sequence and the amino acid sequence of humanized CDl 9 V _H are shown below. Bold marks indicate the mutation (ggc) coding G.

The amino acid sequence of the humanized CD19 scFv (V _L -linker-V _H ) is shown below.

Example 2. Humanized CD 19-CAR Sequences

The scheme of Humanized CD 19-CAR construct is shown in FIG. 2. A lenti viral vector with EF 1 a promoter was used for cloning of humanized scFv CAR sequences.

The following nucleotide sequence and amino acid sequence show CD8 leader- humanized CD 19 ScFv-CD8 hinge-TM CD8-41BB-CD3 zeta of the present invention. The CAR structure includes human CD8 signaling peptide, humanized CD 19 scFv (Vt-Linker- V _H ), human CD8 hinge, human CD8 trans-membrane, human 4 IBB co-stimulatory domain, and human CD3 zeta activation domais (FIG. 2).

<CD8 leader> <Humanized CD19 (VL-linker-VH), clone 11 scFv>

See Example 1 for nucleic acid sequences and amino acid sequences.

<CD8 hinge>

Translated amino-acid sequence of humanized CD 19-4- 1 BB-CD3-C AR protein (see FIG. 2 for construct structure), mutation in CDR1 V _H is shown in bold and underlined.

Example 3. Generation of CAR lentivirus

DNAs encoding the CARs were synthesized and subcloned into a third-generation lenti viral vector with EFla promoter. All CAR lenti viral constructs were sequenced in both directions to confirm CAR sequence and used for lentivirus production. Ten million

HEK293FT cells (Thermo Fisher) were seeded into T75 flasks and cultured overnight, then transfected with the pPACKHl Lentivector Packaging mix ( System Biosciences, Palo Alto, CA) and 10 mg of each lenti viral vector using the CalPhos Transfection Kit (Takara, Mountain View, CA). The next day the medium was replaced with fresh medium, and 48 h later the lenti virus-containing medium was collected. The medium was cleared of cell debris by centrifugation at 2100 g for 30 min. The virus particles were collected by centrifugation at 112,000 g for 100 min, suspended in AIM V medium, aliquoted and frozen at -80 °C. The titers of the virus preparati ons were determi ned by quanti tati ve RT-PCR using the Lenti -X qRT-PCR kit ( Takara ) according to the manufacturer’s protocol and the 7900HT thermal cycler (Thermo Fisher). The lentiviral titers were >1x10 ⁸ pfu/ml.

Example 4. Generation and expansion of CAR-T cells

PBMC were suspended at 1 x 10 ⁶ cells/ml in AIM V-AlbuMAX medium ( Thermo Fisher ) containing 10% FBS and 300 U/ml IL-2 ( Thermo Fisher ), mixed with an equal number (1 : 1 ratio) of CD3/CD28 Dynabeads ( Thermo Fisher ), and cultured in non-treated 24-well plates (0.5 ml per well). At 24 and 48 hours, lenti virus was added to the cultures at a multiplicity of infection (MOI) of 5, along with 1 ml of TransPlus transduction enhancer ( AlStem ). As the T cells proliferated over the next two weeks, the cells were counted every 2- 3 days and fresh medium with 300 U/ml IL-2 was added to the cultures to maintain the cell density at 1-3 x 10 ⁶ cells/ml.

Example 5. Flow cytometry

To measure CAR expression, 0.5 million cells were suspended in 100 ml of buffer (PBS containing 0.5% BSA) and incubated on ice with 1 ml of human serum ( Jackson Immunoresearch, West Grove, PA) for 10 min. Then 1 ml of allophycocyanin (APC)-labeled anti-CD3 ( eBioscience , San Diego, CA), and 2 ml of either phycoerythrin (PE)-labeled Goat anti-mouse or anti-human F(ab) ₂ or isotype control antibodies were added, and the cells were incubated on ice for 30 min. The cells were rinsed with 3 ml of buffer, then suspended in buffer and acquired on a FACSCalibur (BD Biosciences ). Cells were analyzed for CD3 staining versus anti F(Ab)2 staining or isotype control staining.

Example 6. Humanized CD19-CAR-T cells expressed CAR

We designed humanized CD19-CAR-T cells with humanized CD19-CAR construct shown in FIG. 2. We used Mock ScFv with unrelated ScFv (intracellular protein scFv) to generate Mock-CAR-T cells as a negative control.

After transduction of lentiviral humanized CD 19 CAR into T cells, humanized CD 19- CAR construct was detected by FACS analysis with goat-anti-human F(ab)2 antibody by FACS (FIG. 4).

Example 7. Humanized CD19-CAR-T cells killed Hela-CD19 cells but not Hela cells.

We incubated humanized effector CD l 9-CAR-T cells of the present invention with target Hela-CD19 target cells and also Hela (CD 19-negative) control cells and performed real-time cytotoxicity assay (RTCA), as described in [4],

Humanized CDl 9-CAR-T cells specifically killed Hela-CD19 cells as the number of target cell (cell index) significantly decreased by CAR-T cells versus T cells and Mock CAR- T cells (Figure 5, upper panel). Humanized CDl 9-CAR-T cells did not kill CD 19-negative Hela cells, and there was no significant difference between T cells, Mock CAR-T cells and CAR-T cells in killing CD 19-negative Hela cells (FIG. 5, lower panel). This demonstrate high specificity of humanized CDl 9-CAR-T cells to target CD 19 antigen and to kill only CD19-positive cells but not CD19-negative Hela cells (FIG. 5).

Example 8. Humanized CD19 CAR-T cells secreted IFN-gamma against target Hela- CD19 cells.

We collected supernatant after co-incubation of humanized CD19-CAR-T cells and target Hela-CD 19 and performed IFN-gamma assay, as described [5], Humanized CD 19- CAR-T cells secreted IFN-gamma with Hela-CD 19 significantly higher than that secreted by T cells and Mock CAR-T cells (FIG. 6). The level of IFN-gamma secreted by humanized CDl 9-CAR-T cells was significantly higher than by mouse CD19-CAR-T cells (p<0.05) suggesting higher activity of humanized CD 19-CAR-T cells versus CD 19-positive target cells than mouse CD19-CAR-T cells.

Example 9. Humanized CD19-CAR-T cells decreased Raji xenograft tumor growth in vivo

We injected Raji-luciferase-positive cells into NSG-mice intravenously and then next day injected 1x10 ⁷ humanized CD19-CAR-T cells, as described in [4] We used IVIS system to detect Raji xenograft tumor cell growth by imaging. Humanized CD 19-CAR-T cells significantly decreased Raji xenograft growth compared to control PBS and Mock-CAR-T cells (FIG. 7). The decreased Raji tumor growth was more pronounced with humanized CAR-T cells than with mouse CD 19-CAR-T cells (FIG. 7).

The quantification of imaging at day 14 is shown in FIG. 8, which shows that both mouse and humanized CD 19-CAR-T cells significantly decrease the imaging signal-total flux in photons/ sec.

Example 10. Humanized CD19-CAR-T cells significantly prolonged survival of mice in Raji xenograft model over mice treated with mouse CD19-CAR-T cells.

The Kaplan-Meier survival curve shows a significant increase of survival in mice after injecting mice with humanized CD19-CAR-T cells versus mouse CD 19-CAR-T cell or control Mock-Car-T cells (FIG. 9). The results demonstrate increased efficacy of humanized CD19-CAR-T cells compared with mouse CD19-CAR-T cells.

Example 11. Humanized CD19 CAR with a mutation in CDR1 of VH prolonged survival of mice compared with non-mutated humanized CD19 CAR

We compared humanized CD 19-CAR with a V27G mutation in CDR1 of V _H (Clone 1 1) with another humanized CD19-CAR without a mutation in CDR1. The“huCDl 9 no mutation” shown in FIG. 10 is a humanized antibody from mouse clone FMC63 without a mutation in CDR regions. The“huCD19 no mutation” and Clone 11 have the same V _L sequence, but different frame sequence of V _H . In general, antigen binding activity is affected by CDR regions, and not by framework regions.

The results show prolonged survival in mice treated with humanized CD 19-CAR having a V27G mutation in VH than mice treated with humanized CD19-CAR-T cells without a mutation. Example 12. CD19-CAR-T cells were detected by FACS after injection into mice.

Seven days after injecting humanized CD19-CAR-T cells or mouse CD19-CAR-T cells into mice having Raji xenograft tumor cells, mice blood was collected to test the presence of CD19-CAR-T cells. Rabbit anti-FMC63 antibody was used to detect mouse CD19-CAR-T cells. Goat anti-human (Fab’2) antibody (Promab Biotechnologies, Inc.) was used to detect humanized CD19-CAR-T cells.

The results are shown in FIG. 11. Both humanized CD19-CAR-T cells and mouse CD19-CAR-T cells were readily detected after 7 days injecting into mice. The results suggest persistence of CAR-T cells in vivo, which is consistent with significantly decreased Raj i xenograft tumor growth.

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