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 SequenceListing.txt with a creation date of August 13, 2021, and a size of 27 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 BCMA antibody and humanized BCMA-CAR- T Cells (PMC309 with CD28 domain and PMC750 with 41BB domain) specifically decreasing multiple myeloma tumor growth, which are useful in the field of adoptive immunity gene therapy for tumors.

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, 2].

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 coactivation domains: (i) CD28, (ii) CD137 (4-1BB), CD27, or other co-stimulatory domains, in tandem with an activation CD3-zeta domain. (Figure 1) [2, 3], The evolution of CARs went from first generation (with no co-stimulation domains) to second generation (with one costimulation domain) to third generation CAR (with several co-stimulation 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. BCMA

B cell maturation antigen (BCMA) is a cell surface receptor, also known as CD269 and tumor necrosis factor receptor superfamily member 17 (TNFRSF17), that is encoded by TNFRSF17 gene. This receptor is expressed mainly in mature B lymphocytes and in most cases overexpressed in multiple myeloma (MM) [4], Current therapies to target BCMA in MM include monoclonal antibodies, bi-specific antibodies and T cellular immunotherapies, CAR-T therapies [4], [5],

BCMA structure and signaling

The human BCMA protein consists of 184 amino-acids: 1-54-extracellular domain; 55- 77-transmembrane domain; 78-184-cytoplasmic domain. The amino-acid sequence of BCMA is shown on Figure 2. BCMA lacks signaling peptide and resembles other receptors BAFF Receptor and transmembrane activator and cyclophilin ligand interactor and calcium modulator (TACT) [5], These receptors play major role in B cell maturation and differentiation into plasma cells. Their ligands include BAFF and APRIL which expression is increase in MM patients [5], Monoclonal antibodies target receptor-ligand interactions, and CAR-T cell therapy binds BCMA and kill MM cells. BCMA also interacts with TRAF1,2,3,5 and 6.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The structures of CAR. The left panel shows the structure of first generation (no costimulatory domains). The middle panel shows the structure of the second generation (one costimulatory domain CD28 or 4-BB). The right panel shows the third generation of CAR (two or more co-stimulatory domains). [3]

Figure 2. The amino-acid sequence of BCMA protein (SEQ ID NO: 1). Extracellular domain is underlined.

Figure 3. The structure of humanized BCMA CAR construct. The second generation BCMA- CAR has either CD28 as a co-stimulatory domain under EFl-promoter (upper panel, e.g., CAR- PMC309), or 41BB as a co-stimulatory domain under MNDU3 promoter (lower panel, e.g., CAR-PMC750).

Figures 4A-4B. Humanized BCMA-CAR-T cells killed CHO-BCMA cells but not CHO cells. XCelligence Real-time cytotoxicity assay was used for detection of humanized BCMA-CAR-T cell cytotoxicity. Normalized cell index is shown on Y-axis, and time is shown on X-axis.

Figure 4A: CHO-BCMA target cells. Figure 4B: CHO target cells. From top to bottom on the right, Mock CAR-T cells, Humanized BCMA CAR-T cells, T cells and target cells are shown as effector cells.

Figure 5. Humanized BCMA-CAR-T cells secreted high level of IFN-gamma with CHO- BCMA-positive cells. p<0.05, IFN-gamma in CHO-BCMA cells versus T and Mock CAR-T cells.

Figures 6A and 6B. FACS with anti-mouse F(ab)’2 (mF AB) (Figure 6A) and fluorescently labelled BCMA protein (Figure 6B) on hBCMA-CAR-T cells (PMC750) showed high percent of CARpositive cells during 9 days of expansion.

Figures 7A and 7B. RTCA (real-time cytotoxicity assay) demonstrate effective and specific killing of CHO-BCMA cells (7 A), but not CHO-BCMA (7B) by PMC750 CAR-T cells.

Figure 8. IFN-gamma secretion by humanized BCMA PMC750 and mouse BCMA CAR-T cells in CHO-BCMA and Hela-BCMA cells.

Figures 9A-9B. Humanized BCMA-CAR-T cells significantly decreased RPMI8226 xenograft tumor growth. *p<0.001, BCMA-CAR-T cells (PMC750) vs Mock (PBS-control).

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 "extracellular domain capable of binding 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.

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, “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 "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 skilled 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 BCMA scFv starting from heavy and light chain variable regions of a mouse monoclonal antibody, clone 4C8A (W02019/195017). Mouse 4C8A antibody exhibits strong and selective binding to human BCMA. Humanized BCMA antibody of the present invention also exhibits strong and selective binding to human BCMA, but with less immunogenicity to human.

The inventors have generated CAR-T cells based on humanized BCMA ScFv sequence specifically targeting BCMA. The inventors have produced humanized BCMA-CAR-T cells to target cancer cells overexpressing BCMA tumor antigen. The humanized BCMA-CAR-T cells of the present invention secreted high level of cytokines against multiple myeloma cancer cells and killed CHO-BCMA-positive target cells but not control CHO cells.

The present invention is directed to a humanized monoclonal anti-human BCMA antibody or an antigen-binding fragment thereof (e.g., Fab, (Fab)2, scFv), comprising humanized VH having the amino acid of SEQ ID NO: 4 and humanized VL having the amino acid of SEQ ID NO: 5, respectively. In one embodiment, the humanized anti-human BCMA antibody is a single-chain variable fragment (scFv). The scFv can be Vu-linker-Vr or Vr-linker- VH.

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 BCMA (the present invention), (ii) a transmembrane domain, (iii) at least one co-stimulatory domains, and (iv) an activating domain.

In some embodiments, humanized BCMA CAR structures are shown in FIG. 3.

In one embodiment, the co-stimulatory domain is selected from the group consisting of CD28, 4- IBB, GITR, ICOS-1, CD27, OX-40 and DAP 10 domains. A preferred the co- stimulatory domain is CD28 or 4-1BB. A preferred activating domain is CD3-zeta (CD3 Z or CD3Q.

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 P chain, a CD3 zeta chain, CD28, CD3s., CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, 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 BCMA-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 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 (including an oncoretrovirus vector, a lentivirus 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 activated. The activation 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, release 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 humanized BCMA-ScFv-CD28/41-BB-CD3-CAR-T (BCMA-CAR-T) cells against multiple myeloma cells (MM). BCMA-CAR-T cells of the present invention secrete high levels of cytokines. BCMA-CAR-T cells are positive by LDH cytotoxicity assay and by cytotoxicity assay with CHO-BCMA cells but not by CHO cells, which indicates specific killing activity of CAR-T cells against target cancer cells with their cytotoxic activity against tumor or viral antigens.

The advantages of the humanized BCMA -ScFv of the present invention include less immunogenicity to humans because it has human sequences in ScFv, Thus, the BCMA antibody of the present invention is highly potent and advantageous as therapeutic agents in many clinical applications.

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

Humanized BCMA-CAR-T cells using the present humanized BCMA ScFv can be effectively used to target BCMA antigen in BCMA-positive cancer cell lines.

Humanized BCMA-CAR-T cells can be used in combination with different chemotherapy: checkpoint inhibitors; targeted therapies, small molecule inhibitors, antibodies. Humanized BCMA-CAR-T cells can be used clinically for BCMA-positive cancer cells.

Modifications of co-activation domains: CD28, 4- IBB and others can be used to increase its efficacy. Tag-conjugated humanized BCMA scFv can be used for CAR generation.

Humanized BCMA-CAR-T cells can be used with different safety switches: t-EGFR, RQR (Rituximab-CD34-Rituximab) and other.

Third generation CAR-T or other co-activation signaling domains can be used for the same humanized BCMA-scFv inside CAR.

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

The humanized BCMA-CAR-T cells can be used against cancer stem cells that are most resistant against chemotherapy and form aggressive tumors.

Humanized BCMA ScFv or humanized BCMA VH and VL can be used for generation of BCMA bispecific antibodies with another antibody (for example, CD3 ScFv).

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 BCMA-ScFv-CAR construct (CAR-PMC309) under EFl promoter inside lentiviral vector cloned into lentiviral vector. Lentiviral CAR construct contains the humanized BCMA ScFv-CD28-CD3zeta insert - between the Xba I and Eco RI cloning sites. The inventors also generated BCMA-ScFv-41BB-CD3 construct (CAR-PMC750) with CAR under MNDU3 promoter for higher expression of humanized BCMA-CAR (Figure 3).

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.

Materials and Methods

Example 1. Lentiviral CAR Construct

The codon optimized sequence of humanized BCMA ScFv was synthesized in IDT as a Gblock, and sub-cloned into second generation CAR sequence with either CD28 or 4- IBB costimulatory domains and CD3 activation domain. Mock CAR-T cells with extracellular TF tag-CD28-CD3 CAR-T cells were used as Mock CAR-T cells. Example 2. Lentivirus generation

2.5xl0 ⁷ HEK293FT cells (Thermo Fisher) were seeded on 0.01% gelatin-coated 15 cm plates and cultured overnight in DMEM, 2% FBS, Ixpen/strep. The cells were transfected with 10 pg of the CAR lentiviral vector and the pPACKHl Lentivector Packaging mix (System Biosciences, Palo Alto, CA) using the NanoFect transfection NF 100 agent (Alstem). The next day the medium was replaced with fresh medium, and after 48 hours the medium with lentiviral particles was collected. The medium was cleared of cell debris by centrifugation at 2100 g for 30 min. The virus particles were concentrated by ultracentrifugation at 112,000 g for 60 min at 4°C using a SW28.1 rotor, resuspended in serum-free DMEM medium, and frozen in several aliquot vials at -80 °C.

Example 3. CAR-T cells

PBMC were suspended at 1 x 10 ⁶ cells/ml in AIM V-AlbuMAX medium (Thermo Fisher) containing 10% FBS and 10 ng/ml IL-2 (Thermo Fisher) and activated by mixing with an equal number of CD3/CD28 Dynabeads (Thermo Fisher) in non-treated 24-well plates (0.5 ml per well). At 24 and 48 hours, lentivirus was added to the cultures at a multiplicity of infection (MOI) of 5-10. The T and CAR-T cells proliferated over 10-12 days with medium changed every 3 days to maintain the cell density at 1-2 x 10 ⁶ cells/ml.

Example 4. Flow cytometry (FACS)

First, 0.25 million cells were suspended in lOOpl of buffer (PBS containing 2 mM EDTA pH 8 and 0.5% BSA) and incubated on ice with Ipl of human serum for 10 min. The diluted primary antibody was used with cells for 30 min at 4°C, and then after washing the biotin- conjugated goat anti-mouse F(ab)’2 was added with CD3- allophycocyanin (APC)-conjugated mouse anti-human CD3 antibody and PE-conjugated streptavidin at 1 : 100 dilution and incubated for 30 min at 4°C. The cells were rinsed with 3 ml of washing buffer, then stained for 10 min with 7-AAD, suspended in the FACS buffer and analyzed on a FACS Calibur (BD Biosciences). Cells were gated first for light scatter versus 7-AAD staining, then the 7-AAD- live gated cells were plotted for anti-CD3 staining versus CAR+ staining with anti- (Fab)2 antibodies. Example 5. Real Time Cytotoxicity Assay (RTCA)

Adherent target cells (1 x 10 ⁴ cells per well) were seeded into 96-well E-plates (Acea Biosciences, San Diego, CA) using the impedance-based real-time cell analysis (RTCA) xCELLigence system (Acea Biosciences). The next day, the medium was removed and replaced with AIM V-AlbuMAX medium containing 10% FBS ± 1 x 10 ⁵ effector cells in triplicate (CAR-T cells or non-transduced T cells). The cells were monitored for another 24-48 hours with the RTCA system, and impedance was plotted over time. Cytolysis was calculated as (impedance of target cells without effector cells minus impedance of target cells with effector cells) x 100 /impedance of target cells without effector cells.

Example 6. IFN-gamma secretion assay

Non-adherent target cells were cultured with the effector cells (CAR-T cells or nontransduced T cells) at a 1 : 1 ratio (1 x 10 ⁴ cells each) in U-bottom 96-well plates with 200pl of AIM V-AlbuMAX medium containing 10% FBS, in triplicate. After 16 hours, the top 150 pl of medium was transferred to V-bottom 96-well plates and centrifuged at 300 g for 5 min to pellet any residual cells. The top 120pl of supernatant was transferred to a new 96-well plate and analyzed by ELISA for human IFN-y levels using a kit from R&D Systems (Minneapolis, MN) according to the manufacturer’s protocol. The supernatant after RTCA with adherent target cells was collected and analyzed as above.

Example 7. NSG mouse tumor xenograft model and imaging

Six-weeks old male NSG mice (Jackson Laboratories, Bar Harbor, ME) were housed in accordance with the Institutional Animal Care and Use Committee (IACUC). Each mouse was injected subcutaneously on day 0 with lOOpl of 1.5 x 10 ⁶ MMlS-luciferase positive cells in sterile serum free medium. Next day 1 x 10 ⁷ CAR-T cells in serum-free medium were injected intravenously. Imaging was done after luciferin injection using Xenogen Ivis System. Quantification was done by measuring bioluminescence (BLI) in photons/sec signals. Kaplan- Myer survival curve was plotted with GraphPad Prism software based on mice survival data.

Example 8. Statistical analysis.

Data were analyzed with Prism software (GraphPad, San Diego, CA). Comparisons between two groups were performed by unpaired Student’s t-test; comparisons between multiple groups were done with one or two-way ANOVA followed by Sidak or Dunnett’s tests. The p- value <0.05 was considered significant.

Sequences Example 9. Humanized BCMA VH, VL and scFv SEQUENCES

The BCMA scFv was obtained by sequencing hybridoma clones positive for BCMA.

The structure of humanized BCMA (PMC309) scFv is: VH-linker-VL.

The nucleotide sequence of humanized BCMA PMC309 ScFv is shown below: VH is bolded; VL is underlined, in between linker is italicized. caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtg agctgcaaagcgagcggctatacctttaccagctatgtgatgcattgggtgcgccaggcg ccgggccagggcctggaatggatgggctatattattccgtataacgatgcgaccaaatat aacgaaaaatttaaaggccgcgtgaccattaccgcggataaaagcaccagcaccgcgtat atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcgcgcgctataac tatgatggctattttgatgtgtggggccagggcaccctggtgaccgtgagcagcggcggc ggc ggc a gc ggc ggc ggc ggc a gc ggc ggc ggc ggc a gcgatgtggtgatgacccagagc ccggcgtttctgagcgtgaccccgggcgaaaaagtgaccattacctgccgcgcgagccag agcattagcgattatctgcattggtatcagcagaaaccggatcaggcgccgaaactgctg attaaatatgcgagccagagcattagcggcgtgccgagccgctttagcggcagcggcagc ggcaccgattttacctttaccattagcagcctggaagcggaagatgcggcgacctattat tgccagaacggccatagctttccgccgacctttggcggcggcaccaaagtggaaattaaa (SEQ ID NO: 2)

Humanized BCMA (PMC309) scFv, amino acid sequence is shown below (SEQ ID NO: 3)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYND

ATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGT

LVTVSSGGGG GGGG GGGG5DVVMTOSPAFLSVTPGEKVTITCRASOSISDYLHWYOQK

PDOAPKLLIKYASOSISGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNGHSFPP TFGGGT KVEIK

BCMA (PMC309), VH, amino acid sequence (SEQ ID NO: 4):

QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYND

ATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARYNYDGYFDVWGQGT

LVTVSS

BCMA (PMC309) VL, amino acid sequence (SEQ ID NO: 5):

DVVMTOSPAFLSVTPGEKVTITCRASOSISDYLHWYOOKPDQAPKLLIKYASOSISG VPSRF SGSGSGTDFTFTISSLEAEDAATYYCQNGHSFPPTFGGGTKVEIK

The linker amino sequence is shown below (SEQ ID NO: 6)

GGGGSGGGGSGGGGS

Example 10A. Humanized BCMA-CAR Sequences (CAR-PMC309)

The scheme of Humanized (PMC309) BCMA-CAR construct is shown on Figure 3. Lentiviral vector with EFla promoter was used for cloning of humanized scFv CAR sequences.

The following nucleotide sequence shows human CD8 signaling peptide, humanized BCMA scFv (Vu-Linker-VL), CD8 hinge, CD28 transmembrane, co-stimulating domain CD 28, activation domain CD3 zeta (Figure 3, upper panel).

<CD8 leader>

Nucleotide sequence, SEQ ID NO: 7

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCC AGGCCG

Amino acid sequence, SEQ ID NO: 8

MALPVTALLLPLALLLHAARP

<Nhe I site>

Nucleotide sequence gctagc

Amino Acid Sequence

AS

<Humanized BCMA (PMC309) scFv>

See Example 9.

<XhoI restriction site>

Nucleotide sequence

CTCGAG

Amino acid sequence

LE

<CD8 hinge>

Nucleotide sequence, SEQ ID NO: 9 AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTC

GCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGC

ACACGAGGGGGCTGGACTTCGCCAGTGAT

Amino acid sequence, SEQ ID NO: 10

KPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD

<Spacer>

Nucleotide sequence aagccc

Amino Acid sequence

KP

<CD28 TM/co-stimulating>

Nucleotide sequence, SEQ ID NO: 11

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACA

GTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGA CTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCT ATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC

Amino acid sequence, SEQ ID NO: 12

FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY A

PPRDFAAYRS

<CD3 zeta>

Nucleotide sequence, SEQ ID NO: 13

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC

AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG

AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTC

AGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGA GATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAG GGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCC CCCTCGCTAAtag

Amino acid sequence, SEQ ID NO: 14

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

<EcoRI restriction site> gaattc

Nucleotide sequence of humanized BCMA-CAR protein (CAR-PMC309), SEQ ID NO: 15 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg ccggctagccaggtgcagctggtgcagagc ggcgcggaagtgaaaaaaccgggcagcagcgtgaaagtgagctgcaaagcgagcggctat acctttaccagctatgtgatgcattgggt gcgccaggcgccgggccagggcctggaatggatgggctatattattccgtataacgatgc gaccaaatataacgaaaaatttaaaggccg cgtgaccattaccgcggataaaagcaccagcaccgcgtatatggaactgagcagcctgcg cagcgaagataccgcggtgtattattgcgc gcgctataactatgatggctattttgatgtgtggggccagggcaccctggtgaccgtgag cagcggcggcggcggcagcggcggcggc ggcagcggcggcggcggcagcgatgtggtgatgacccagagcccggcgtttctgagcgtg accccgggcgaaaaagtgaccattacct gccgcgcgagccagagcattagcgattatctgcattggtatcagcagaaaccggatcagg cgccgaaactgctgattaaatatgcgagcc agagcattagcggcgtgccgagccgctttagcggcagcggcagcggcaccgattttacct ttaccattagcagcctggaagcggaagatg cggcgacctattattgccagaacggccatagctttccgccgacctttggcggcggcacca aagtggaaattaaactcgagaagcccacca cgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccc tgcgcccagaggcgagccggccagcg gcggggggcgcagtgcacacgagggggctggacttcgccagtgataagcccttttgggtg ctggtggtggttggtggagtcctggcttgc tatagcttgctagtaacagtggcctttattattttctgggtgaggagtaagaggagcagg ctcctgcacagtgactacatgaacatgactcccc gccgccccgggcccacccgcaagcattaccagccctatgccccaccacgcgacttcgcag cctatcgctccagagtgaagttcagcagg agcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatcta ggacgaagagaggagtacgatgttttgga caagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctca ggaaggcctgtacaatgaactgcaga aagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggca aggggcacgatggcctttaccagggtc tcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgct aa

Amino acid sequence of humanized BCMA-CAR protein (CAR-PMC309), SEQ ID NO: 16

MALPVTALLLPLALLLHAARPASQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVM H WVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAV YYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPG

EKVTITCRASQSISDYLHWYQQKPDQAPKLLIKYASQSISGVPSRFSGSGSGTDFTF TISSL

EAEDAATYYCQNGHSFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSLRP EASR

PAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSD Y

MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGR REEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRG

KGHDGLYQGLSTATKDTYDALHMQALPPR

Example 10B. Humanized BCMA-CAR Sequences (CAR-PMC750)

Another CAR was prepared similar to the protocols of Example 2A with the same scFv, except with 4 IBB domain as a stimulating domain inside lentiviral vector with Kan resistance gene (FIG. 3, lower panel, CAR-PMC750).

The amino-acid sequence of hBCMA-41BB-CD3 CAR (CAR-PMC750) is shown below as SEQ ID NO: 17, with 41BB shown bold.

MALPVTALLLPLALLLHAARPASQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYVM H

WVRQAPGQGLEWMGYIIPYNDATKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTA V

YYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPG

EKVTITCRASQSISDYLHWYQQKPDQAPKLLIKYASQSISGVPSRFSGSGSGTDFTF TISSL

EAEDAATYYCQNGHSFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSLRP EASR

PAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELN

LGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR

RGKGHDGLYQGLSTATKDTYDALHMQALPPR

Results

Example 11. Humanized BCMA-CAR-T cells killed CHO-BCMA cells but not CHO cells.

The lentivirus was prepared using 293 S cells as described in [6], We transduced T cells with Humanized BCMA-CAR lentivirus, CAR-T cells were expanded and expressed BCMA scFv, which was detected with BCMA recombinant protein (as described [6]). Then, we incubated humanized BCMA-CAR-T cells with target CHO-BCMA target cells and also with CHO (BCMA-negative) control cells. Humanized BCMA-CAR-T cells specifically killed CHO- BCMA cells (Figure 4A) but not CHO cells (Figure 4B). This demonstrate high specificity of humanized BCMA-CAR-T cells to targeting BCMA antigen and killing BCMA-positive cells.

Example 12. Humanized CAR-T cells secrete IFN-gamma against target CHO-BCMA cells significantly.

We collected supernatant after co-incubation of humanized BCMA-CAR-T cells and target CHO-BCMA cells and performed IFN-gamma assay. BCMA-CAR-T cells secreted high level of IFN-gamma with CHO-BCMA cells (Figure 5). Low secretion of IFN-gamma was observed with control CHO cells (Figure 5). This confirms the specificity of humanized BCMA- CAR-T cells. Humanized BCMA CAR-T cells also secreted higher levels of IFN-gamma against RPMI8226 cells compared with BCMA-negative K562 cells (datanot shown).

Example 13. Humanized BCMA-41BB-CD3 CAR-T cells (PMC750) expressed high percent of CAR-positive cells, killed BCMA-positive target cells and secreted high level of IFN-gamma.

We re-cloned humanized BCMA with 4 IBB co-stimulatory domain and MNDU3 promoter to have higher persistency of CAR-T cells, after transduction, CAR-T cells had high percent of BCMA ScFv-positive cells (Figures 6A-6B). We detected >70% CAR-positive cells at day 9 after expansion that was detected with both anti-mouse F(ab)2 and recombinant fluorescently labelled BCMA protein (Figures 6A-6B).

We performed cytotoxicity assay using PMC750 h BCMA-CAR-T cells as effector cells and CHO-BCMA cells as target cells. hBCMA-41BB-CD3 CAR-T cells effectively killed CHO- BCMA cells (Figure 7 A) and did not kill control BCMA-negative CHO-CS1 cells, while CS1- CAR-T cells killed (Figure 7B).

We detected high level of IFN-gamma secreted by PMC750 hBCMA-CAR-T cells against CHO and Hela-BCMA-positive cells, which were higher than IFN-gamma level secreted by mouse BCMA-CAR-T cells (Figure 8).

Example 14. Humanized BCMA-CAR-T cells significantly decreased RPMI8226 xenograft tumor growth in mouse model in vivo.

Multiple myeloma RPMI8226-luciferase positive cells were injected intravenously into NSG mice (2xl0 ⁶ cells/mice), and then next day humanized BCMA-CAR-T cells (PMC750) were injected by i.v. (IxlO ⁷ CAR-T cells/mice). The imaging with luciferin was performed to detect tumor growth (Figure 9A). Humanized BCMA-CAR-T cells significantly decreased RPMI8226 tumor growth in mice, p<0.001 (Figure 9B). No behavior or visual changes were observed during the study.

References

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