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 December 17, 2020, and a size of 22.0 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 a humanized BCMA antibody and BCMA-CAR-T Cells. The humanized BCMA-CAR-T cells specifically decrease multiple myeloma tumor growth, and they 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”)[l, 3]

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) CD137 (4-1BB), CD27, or other co stimulatory 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 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) [3] Current therapies to target BCMA in MM include monoclonal antibodies, bi-specific antibodies and T cellular immunotherapies, CAR-T therapies [3], [4]

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 (TACI) [4] 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 [4] 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. This invention is based on humanized BCMA-CAR-T cells targeting BCMA in MM. The advantage of humanized BCMA-CAR-T cells it has humanized BCMA scFv that is less immunogenic than mouse ScFv to humans because it has human sequences in scFv.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The structures of CAR. On the left panel: the structure of first generation (no costimulatory domains). On the middle panel-second generation with one co-stimulation domain (CD28 or 4-BB). On the right panel- third generation of CAR (two or several co stimulation domains) are shown [6]

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. This is second generation of CAR.

Figure 4. Humanized BCMA-CAR-T cells killed CHO-BCMA cells but not CHO cells. Figure 5. Humanized BCMA-CAR-T cells secreted high level of IFN-gamma against CHO-BCMA-positive cells. p<0.05, IFN-gamma in CHO-BCMA cells versus T and Mock CAR-T cells.

Figure 6. Humanized BCMA-CAR-T cells secreted high level of IFN-gamma against multiple myeloma cells but not against BCMA-negative K562 control cells. p<0.05, IFN- gamma in multiple myeloma cells versus T and Mock-CAR-T cells; and versus IFN-gamma levels in K562 cells.

Figure 7A. Humanized BCMA-CAR-T cells significantly decreased RPMI8226 xenograft tumor growth.

Figure 7B. Humanized BCMA-CAR-T cells did not decrease mouse body weight.

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, “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 skilled in the art. As used herein, a "tumor antigen" means a biological molecule having antigenicity, expression of which causes cancer.

The present invention is directed to a humanized monoclonal anti-human BCMA antibody clone (PM 308), obtained by sequencing and humanizing mouse monoclonal anti- BMCA antibody (hybridoma clone 4C8A, W02019/195017). The humanized anti-human BCMA antibody comprises humanized VH having the amino acid of SEQ ID NO: 4 and humanized VL having the amino acid of SEQ ID NO: 5. In one embodiment, the humanized anti-human BCMA antibody is a single-chain variable fragment (scFv). ScFv can be VH- linker-VL or VL-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 one embodiment, the co-stimulatory domain of CAR is selected from the group consisting of CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAPIO. A preferred the co stimulatory domain is CD28 or 4-1BB.

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, 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 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 (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 CAR-T cells based on a humanized BCMA ScFv sequence specifically targeting BCMA. The inventors have produced humanized BCMA-CAR- T cells to target cancer cells overexpressing BCMA tumor antigen. BCMA-CAR-T cells secreted high levels of cytokines, were positive by cytotoxicity assay with CHO-BCMA cells but not by control 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 human due to humanized BCMA 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 or antibody 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, 4-1BB 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 other 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.

The present BCMA-CAR can be used to generate other types of cells such as BCMA- CAR-natural killer (NK) cells, BCMA-CAR-macrophages, and other BCMA-CAR hematopoietic cells, which can target BCMA-positive cancers. The present invention provides T cells, or NK cells, macrophages, or other hematopoietic cells, modified to express the BCMA-CAR.

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 constructs inside lentiviral vector cloned into Xba I and Eco R I sites of lentiviral vector. The lentiviral CAR construct contained the humanized BCMA ScFv-CD28/4-lBB-CD3zeta insert - between the Xba I and Eco RI cloning sites. The CAR was under either Efl or MNDU3 promoter.

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.

Example1. HumanizedBCMA VH andVL andscFvSequences

The BCMA scFv was obtained by sequencing hybridoma clones 4C8A4 and 4C8A10 positive for BCMA. The structure of humanized BCMA (PMC308) scFv is: VH-linker-VL. Linker is G4Sx3.

The bold highlights the nucleotide sequence of humanized BCMA PMC308 ScFv clone: V _H ; the underlined highlights the nucleotide sequence of V _L ; in between (italicized) is the nucleotide sequence encoding a linker. caggtgcagctggtgcagagcggcgcggaagtgaaaaaaccgggcgcgagcgtgaaagtg agctgcaaagcgagcggctatacctttaccagctatgtgatgcattgggtgcgccaggcg ccgggccagggcctggaatggatgggctatattattccgtataacgatgcgaccaaatat aacgaaaaatttaaaggccgcgtgaccatgacccgcgataccagcaccagcaccgtgtat atggaactgagcagcctgcgcagcgaagataccgcggtgtattattgcgcgcgctataac tatgatggctattttgatgtgtggggccagggcaccctggtgaccgtgagcagcggcggc ggcggcagcggcggcggcggcagcggcggcggcggcagcgatgtggtgatgacccagagc ccggcgtttctgagcgtgaccccgggcgaaaaagtgaccattacctgccgcgcgagccag agcattagcgattatctgcattggtatcagcagaaaccggatcaggcgccgaaactgctg attaaatatgcgagccagagcattagcggcgtgccgagccgctttagcggcagcggcagc ggcaccgattttacctttaccattagcagcctggaagcggaagatgcggcgacctattat tgccagaacggccatagctttccgccgacctttggcggcggcaccaaagtggaaattaaa (SEQ ID NO: 2) Humanized BCMA (PMC308) scFv Protein (SEQ ID NO: 3):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAP GQGLEWMGYIIPYNDATKYNEKFKGRVTMTRDTSTSTVYME LSSLRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSS GGGGSG YOOKPDOAPKLLIKYASOSISGVPSRFSGSGSGTDFTFTISSLEAE DAATYY CONGHSFPPTF GGGTKVEIK

In the scFv protein, the bold highlights the amino acid sequence of V _H (SEQ ID NO: 4): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPG QGLEWMGYIIPYNDATKYNEKFKGRVTMTRDTSTSTVYMELSS LRSEDTAVYYCARYNYDGYFDVWGQGTLVTVSS

In the scFv protein, the underlined highlights the amino sequence of V _L (SEQ ID NO: 5): DVVMTQSPAFLSVTPGEKVTITCRASQSISDYLHWYQQKPDQAP KLLIKYASQSISGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQ NGHSFPPTF GGGTKVEIK

The linker sequence is 3xG4S (SEQ ID NO: 6).

GGGGSGGGGSGGGGS

Example 2. Humanized BCMA-CAR Sequences

The scheme of humanized (PMC308) 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 and amino acid sequences show CD8 leader-humanized

BCMA ScFv-CD8 hinge-TM28-CD28/4-lBB-CD3 zeta of the present invention. The CAR structure includes human CD8 signaling peptide (CD8 leader), humanized BCMA scFv (V _H - Linker 3x(G4S) -V _L ), human CD8 hinge, human CD28 transmembrane, co-stimulating domains CD28 or 4- IBB, human CD3 zeta (Figure 3). A CD28 as a co-stimulating domain

The nucleic acid sequence and amino acid sequence of each segment of CD8 leader- BCMA scFv (VH-Linker-VL)-CD8 hinge-CD28 TM-CD28-CD3-zeta (BCMA-CD28 CAR) are shown below.

<CD8 leader>

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCGgctagc (SEQ ID NO: 7)

MALP VT ALLLPL ALLLHAARP AS (SEQ ID NO: 8)

<Humanized BCMA scFv, clone 4C8A>

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

<XhoI restriction site> CTCGAG

<CD8 hinge underlined>

AAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCG

TCGCAGCCCCTGTCCCTGCGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCA

GT GC AC AC GAGGGGGC T GGAC TTCGC C AGT GAT aagccc (SEQ ID NO: 9)

_K P TTTPAPRP PTPAPTIAS QPLSLR PEASRPAAGGAVH TRGLD FAS DKP (SEQ ID NO: 10)

<CD28 TM>

TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAA CAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 11)

FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 12)

<CD28/Co-stimulation domain>

AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGC CGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCG CAGCCTATCGCTCC (SEQ ID NO: 13)

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 14)

<CD3 zeta>

AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAAC

C AGC TC T AT A AC G AGC T C A AT C T AGG AC G A AG AG AGG AGT AC GAT GTT TT GGAC

AAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAA

CCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA

CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC

TTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCA

GGCCCTGCCCCCTCGCTAAtag (SEQ ID NO: 15) RVKF SRS AD AP AY QQGQNQLYNELNLGRREEYD VLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAY SEIGMKGERRRGKGHDGLY QGLSTATKDT YD ALHMQ A LPPR (SEQ ID NO: 16)

<EcoRI restriction site> gaattc Translated amino-acid sequence of humanized BCMA-CAR protein with CD28 as a co stimulating domain is shown below.

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQGLEWMGYIIPYN DATKYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARYNYDGYFDVWGQ GTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPAFLSVTPGEKVTITCRASQSISDYLH WYQQKPDQAPKLLIKYASQSISGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQNGH SFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGL DFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGP TRKHY QP YAPPRDF AAYRSRVKF SRS AD AP AY QQGQNQLYNELNLGRREEYD VLD KRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL YQGLST ATKDTYD ALHMQ ALPPR (SEQ ID NO: 17)

B. 41-BB as a co-stimulating domain

We also generated CAR (PMC709) under MNDU3 promoter using the same humanized BCMA scFv with 4- IBB domain instead of CD28 domain as a co-stimulating domain.

Nucleotide sequence of 4-1BB domain:

AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GT AC A A AC T AC TC A AG AGG A AG AT GGC T GT AGC T GC C G AT TT C C AG A AG A AG A A G A AGG AGG AT GT G A AC T G (SEQ ID NO: 18)

Amino acid sequence of 4- IBB domain:

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 19)

Nucleotide sequence of CAR (4- IBB)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCG CCAGGCCGGCTAGCCAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAA CCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACCTTTACCAGC TATGTGATGCATTGGGTGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATGGGC TATATTATTCCGTATAACGATGCGACCAAATATAACGAAAAATTTAAAGGCCGCG TGACCATGACCCGCGATACCAGCACCAGCACCGTGTATATGGAACTGAGCAGCC TGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCTATAACTATGATGGCTA TTTTGATGTGTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGGCGGCGGCGG

CAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATGTGGTGATGACCCAGA

GCCCGGCGTTTCTGAGCGTGACCCCGGGCGAAAAAGTGACCATTACCTGCCGCG

CGAGCCAGAGCATTAGCGATTATCTGCATTGGTATCAGCAGAAACCGGATCAGG

CGCCGAAACTGCTGATTAAATATGCGAGCCAGAGCATTAGCGGCGTGCCGAGCC

GCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCTTTACCATTAGCAGCCTGGA

AGCGGAAGATGCGGCGACCTATTATTGCCAGAACGGCCATAGCTTTCCGCCGAC

CTTTGGCGGCGGCACCAAAGTGGAAATTAAACTCGAGAAGCCCACCACGACGCC

AGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTG

CGCCCAGAGGCGAGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCT

GGACTTCGCCAGTGATAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTG

GCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAAACGGG

GCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAA

CTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAG

GATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGC

AGGGCC AGAACC AGCTCT AT AACGAGCTC AATCT AGGACGA AGAGAGGAGT ACG

ATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGA

GAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATG

GCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGG

GCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGC

CCTTCACATGCAGGCCCTGCCCCCTCGCTAA (SEQ ID NO: 20)

Amino acid sequence of CAR (4- IBB)

MALPVTALLLPLALLLHAARPASQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYV

MHW VRQ APGQGLEWMGYIIP YND ATK YNEKFKGRVTMTRDT S T S T VYMEL S SLRS

EDTAVYYCARYNYDGYFDVWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPA

FLSVTPGEKVTITCRASQSISDYLHWYQQKPDQAPKLLIKYASQSISGVPSRFSGSG SG

TDFTFTISSLEAEDAATYYCQNGHSFPPTFGGGTKVEIKLEKPTTTPAPRPPTPAPT IAS

QPLSLRPEASRPAAGGAVHTRGLDFASDKPFWVLVVVGGVLACYSLLVTVAFIIFWV

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQ

GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMA

EAY SEIGMKGERRRGKGHDGL Y QGL ST ATKDT YD ALHMQ ALPPR (SEQ ID NO: 21)

Example 3. CAR Lentivirus Production

The inventors generated BCMA CAR constructs inside lentiviral vector cloned into lentiviral vector. BCMA-CD28-CD3 lentiviral CAR construct containing the BCMA ScFv- CD28-CD3zeta insert was prepared under EF1 promoter (PMC308). BCMA-4-1BB-CD3 lentiviral CAR construct containing the BCMA ScFv-4-lBB-CD3zeta insert was prepared under MNDU3 promoter (PMC709). The lenti viruses were generated in 293 T cells and titer was established by RT-PCR or by functional FACS with 293 cells as described [7] Then equal dose of lentiviruses was used for transduction ofT cells.

Example 4. Peripheral Blood Mononuclear cell (PBMC) Isolation from Whole Blood

Whole blood (Stanford Hospital Blood Center, Stanford, CA) was collected from individual or mixed donors (depending on the amount of blood required) in 10 mL Heparin vacutainers (Becton Dickinson). Approximately 10ml of whole anti-coagulated blood was mixed with sterile phosphate buffered saline (PBS) buffer for a total volume of 20ml in a 50ml conical centrifuge tube (PBS, pH 7.4, is without Ca ⁺² /Mg ⁺² ). The layer of cells containing peripheral blood mononuclear cells (PBMC), seen at the diluted plasma/Ficoll interface was removed very carefully, avoiding any Ficoll, washed twice with PBS, and centrifuged at 200xg for lOmin at room temperature. Cells were counted with a hemocytomter. The PBMC were washed once with CAR-T media (AIM V-AlbuMAX(BSA) (Life Technologies), with 5% AB serum and 1.25 ug/mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U/mL penicillin, and 100 ug/mL streptomycin) and were either used for experiments or were frozen at -80°C for next experiment.

Example 5. T-Cell Activation from PBMC

Freshly isolated PBMC were washed with lxPBS (pH7.4, no Ca ²⁺ /Mg ²⁺ ) and washed once in CAR-T media (AIM V-AlbuMAX(BSA)(Life Technologies), with 5% AB serum and 1.25 pg/mL amphotericin B (Gemini Bioproducts, Woodland, CA), 100 U/mL penicillin, and 100 pg/mL streptomycin), in the absence of human interleukin-2 (huIL-2)(Invitrogen), at a concentration of 5 x 10 ⁵ cells/mL. The cells were resuspended to a final concentration of 5xl0 ⁵ cells/mL in CAR-T medium with 300U/mL huIL2. The PBMC was activated with CD3-CD28 beads with 1:1 bead-to-cell ratio. Desired number of aliquots were dispensed to single wells of a culture plate, and then incubated at 37°C in the presence of CO2 for 24 hours before viral transduction.

Example 6. T-Cell Transduction and Expansion

Following activation of PBMC, the cells were incubated for 24 hr at 37°C, 5% CO2.

To each well of lxlO ⁶ cells, 5xl0 ⁶ lentivirus, and 2 pL/mL of media of Transplus (Alstem, Richmond, CA) (a final dilution of 1 :500) were added. Cells were incubated for an additional 24 hours before repeating addition of virus. Cells were then grown in the continued presence of 300 U/Ml of IL-2 Fresh medium with IL-2 for a period of 12-14 days (total incubation time was dependent on the final umber of CAR-T cells required). Cells concentrations were analyzed every 2-3 days, with media being added at that time to dilute the cell suspension to lxlO ⁶ cells/mL.

Example 7. FACS for Detection of CAR-Positive Cells Vita, please check am! revise!

Cells were washed and suspended in FACS buffer (phosphate-buffered saline (PBS) plus 0.1% sodium azide and 0.4% BSA). Cells were divided them lxlO ⁶ aliquots.

Fc receptors were blocked with normal goat IgG (Life Technologies) in ice for 10 min.

Biotin-labeled polyclonal goat anti-mouse-F(ab)2 antibodies (Life Technologies) were used to detect BCMA ScFv; biotin-labeled normal polyclonal goat IgG antibodies (Life Technologies) were used as an isotype control. (1:200 dilution, reaction volume of 100 mΐ). Cells were incubated at 4°C for 25 minutes and washed once with FACS buffer.

Cells were suspended in FACS buffer and blocked with normal mouse IgG (Invitrogen) by adding 100 mΐ 1 : 1000 diluted normal mouse IgG to each tube. Cells were incubated in ice for 10 min and washed with FACS buffer and re-suspended in 100 mΐ FACs buffer. The cells were then stained with phycoerythrin (PE)-labeled streptavidin (BD Pharmingen, San Diego, CA) and allophycocyanin (APC)-labeled CD3 (eBiocience, San Diego, CA).

Example 8. Cytotoxicity Assay

The Real-time Cytotoxicity Assay (RTCA) was performed using ACEA machine according to manufacturer’s protocol as described [8]

Example 9. Humanized BCMA-CAR-T cells killed multiple myeloma cells and secreted high level of IFN-gamma against BCMA-positive cancer cells.

We designed humanized BCMA-CAR-T cells with humanized BCMA-CAR construct (PMC308) shown in Figure 3. We used Mock scFv with unrelated scFv and generated Mock- CAR-T cells as a negative control. Humanized BCMA-CAR-T cells expressed BCMA scFv as detected by FACS (see Example 7). The results show that 43.7% of BCMA-CAR (PMC308) cells were detected by FACS with a mouse FAB antibody, whereas only 1.79% of control T cells were positively detected. Example 10. Humanized BCMA-CAR-T cells killed CHO-BCMA cells but not CHO cells

We incubated humanized BCMA-CAR-T cells (PMC308) with target CHO-BCMA target cells and also CHO (BCMA-negative) control cells. XCelligence real-time cytotoxicity assay (RTCA) was used for detection of humanized BCMA-CAR-T cell cytotoxicity. In Figure 5, normalized cell index is shown on Y-axis, and time is shown on X- axis. Upper panel: CHO-BCMA target cells on the right: From top to bottom: Mock, T cells, Mock-CAR-T cells and humanized CAR-T cells are shown as effector cells. Lower panel: 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.

The results show that humanized BCMA-CAR-T cells specifically killed CHO- BCMA cells (Figure 4, upper panel) but not CHO cells (Figure 4, lower panel). This demonstrates high specificity of humanized BCMA-CAR-T cells to target BCMA antigen positive target cells and kill BCMA-positive target cells.

Example 11. Humanized CAR-T cells secreted 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). There was no high secretion of IFN-gamma with control CHO cells (not shown). This confirms specificity of humanized BCMA-CAR-T cells and killing cytotoxicity assay.

Example 12. Humanized CAR-T cells secreted high levels of IFN-gamma against BCMA-positive RPMI8226 multiple myeloma cells but not against BCMA-negative leukemia K562 cells

We incubated BCMA-CAR-T cells with multiple myeloma cancer cells RPMI8226 cells and K526 cells and performed ELISA with IFN-gamma using kit from Fisher, according to Fisher’s protocol. Humanized BCMA-CAR-T cells secreted high level of IFN- gamma against BCMA-positive multiple myeloma cancer cells but not against BCMA- negative leukemia K562 cells (Figure 6). The level of killing and secretion of IFN-gamma was significantly higher than with control T and Mock CAR-T cells. This confirms specificity of humanized BCMA-CAR-T cells against hematological BCMA-positive cells.

We also tested BCMA-CAR-T cells (PMC709) which has the same humanized BCMA scFv, but with 4-1BB-CD3 inside lentivirus with MNDU3 promoter. PMC709 BCMA-CAR-T cells also killed CHO-BCMA target cells (data not shown).

Example 13. Humanized BCMA-CAR-T cells significantly decreased RPMI8226 xenograft tumor growth in mouse model in vivo.

Multiple myeloma RPMI8226 cells were injected subcutaneously into NSG mice (lxlO ⁷ cells/mice), and then humanized BCMA-CAR-T cells were injected twice by i.v. (lxlO ⁷ CAR-T cells/mice). Figure 7 A shows humanized BCMA-CAR-T cells significantly decreased RPMI8226 tumor growth in mice. CAR-T cells were injected at day 7 and 20 by i.v 1c10 ^L 7 cells/mice. Bars show average tumor volume +/- standard errors. *p<0.05,

BCMA vs Mock.

Figure 7B shows that mice treated with humanized BCMA-CAR-T cells did not cause a decrease in body weight suggesting that CAR-T cells were not toxic to mice. No behavior or visual changes of mice were observed during the study.

Humanized BCMA-CAR-T were detected in the mouse blood by FACS with BCMA recombinant protein (not shown).

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