Nanobody-based antibodies and car t cells and methods of using the same

Abstract

The present invention relates to the generation of nanobody-based antibodies or nanobody-based CAR T cells that can be as functional as scFv-based antibodies or scFv-based CAR T cells in treating a disease. Single variable domain on a heavy chain (VHH) (also called nanobodies) are used to target domains of antibodies or CARs.

Description

[0001] NANOBODY-BASED ANTIBODIES AND CAR T CELLS AND METHODS OF USING THE SAME

[0002] SEQUENCE LISTING

[0003] A Sequence Listing conforming to the rules of WiPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on February 10, 2025, is entitled " 2541 -22 PCT ST26.xml" and is 286,403 bytes in size.—

[0004] FIELD OF THE INVENTION

[0005] The presently invention generally relates to a chimeric antigen receptor (CAR) or an antibody ligand for targeting cancer cells and tumors as well as immune cells associated with autoimmune diseases. Also provided are therapeutic and diagnostic uses for the same.

[0006] The present invention relates to the generation of nanobody-based antibodies or nanobody- based CAR T cells that can be as functional as scFv-based antibodies or scFv-based CAR T cells in treating a disease. Single variable domain on a heavy chain (VHH) (also called nanobodies) are used to target domains of antibodies or CARs.

[0007] The present invention also relates to a camelid BCMA VHH, CD 19 VHH, Claudin 18.2 VHH and CLL1 and the generation of CAR-T cells comprising these camelid VHHs and method of using the same. Finally, the present invention relates to a humanized BCMA and CD 19 scFv, and the generation of CAR-T cells comprising a humanized BCMA scFv and CD19 scFv.

[0008] BACKGROUND OF THE INVENTION

[0009] Cancers arise when normal cells go awry. Particularly, cancers arise when classical mechanisms required for cell survival are dysregulated to give the cell a proliferative advantage. While many strides have been made in the field of chemotherapy, drug resistance is a challenge that remains unsolved. An alternative, more effective approach presents itself in the guise of harnessing the patient’s own immune system to target the cancer. While B-cell malignancies have been targeted with standard therapy, these classical modes of treatment are often insufficient and accompanied by toxic side effects. The field of immunotherapy has progressed immensely in the past decade and many tumor antigens have been identified. However, these tumor antigens are derived from the patient and are thus poorly immunogenic. As a result, they are unable to surpass the threshold of clinical effectiveness. CAR-T cell therapy has shown much promise in treating hematologic malignancies that have evaded standard chemotherapies and gained resistance. Particularly, CD19-specific CAR-T cell (CD 19 CAR) therapies are extremely effective against B- cell malignancies and have shown remarkable outcomes such as long-tcrin remissions and decreased relapse rates. However, patients suffering from B-ALL, have a relatively high level of relapses within one year of treatment with traditional CD19 CAR, and this relapse can be attributed to antigen escape, CAR persistence, T-cell exhaustion and other underlying factors that together contribute to the need for more effective CAR-T cell treatments.

[0010] Gastric cancers present a significant global health challenge as the fifth most common cancer and the fourth leading cause of cancer death worldwide, with 1,089,103 new cases and 768,793 deaths reported in 2020. Alarmingly, there is a rising incidence of early-onset gastric cancer among young adults under 50. The prognosis for advanced gastric cancers remains dire, with a five-year relative survival rate of only 6%. Traditional molecular approaches to treatment have been hampered by inappropriate biomarker selection, suboptimal trial design, and the spatial and temporal heterogeneity of gastric cancer (Nat Rev Gastroenterol Hepatol 20, 155-170 (2023)). Thus, there is a strong felt need for new, more effective treatment options for gastric cancers.

[0011] Single targeting CD 19 and dual-targeting BCMA-CD19 chimeric antigen receptor (CAR) T-cell therapy has shown promise in the treatment of, for example, autoimmune diseases. All FDA approved CD 19 CAR-T treatments utilize murine derived single-chain fragment variable (scFv), FMC63 clones, comprised of light and heavy chains joined by an immunogenic peptide linker. Neutralizing antibodies against the CAR scFv have been detected and undermine the ability to re- dose CAR-T cells and to cure the disease. There is a need for novel, more effective treatments for autoimmune diseases.

[0012] The present invention provides solutions to the deficiencies in the prior art treatment strategies by aiming at generating more effective CARs in treating diseases in terms of efficacy and safety.

[0013] SUMMARY OF THE INVENTION The present invention provides camelid chimeric antigen receptors (camCARs) targeting hematologic malignancies and autoimmune disorders, compositions and methods of use thereof.

[0014] In one embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide (also called leader sequence), a camelid-derived antigen recognition domain (VHH). a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0015] In one embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a camelid-derived CD19 antigen recognition domain (VHH), a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0016] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a camelid-derived BCMA antigen recognition domain ( VHH), a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0017] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a camelid-derived Claudin 18.2 antigen recognition domain ( VHH), a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0018] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a camelid-derived CLL1 antigen recognition domain (VHH), a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0019] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a humanized BCMA antigen recognition domain (scFv) a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0020] In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a humanized CD 19 antigen recognition domain (scFv) a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain x In another embodiment, the invention provides an engineered chimeric antigen receptor polypeptide comprising: a signal peptide, a humanized BCMA antigen recognition domain (scFv) a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0021] In one embodiment, the present disclosure provides an engineered cell including a chimeric antigen receptor polypeptide comprising a signal peptide, antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a co-stimulatory domain; wherein said antigen recognition domain comprises one of CD 19, BCMA, CD20, CD7 and Claudin 18.2; and at least one enhancer; wherein a high efficiency cleavage site is disposed between the chimeric antigen receptor polypeptide and enhancer. A high efficiency cleavage sites include, but not limited to, P2A, T2A, F2A and E2A. In a further embodiment, the cells comprising the enhancer are expressed in a single open reading frame (ORF) with a CAR under the control of a strong promoter.

[0022] In another embodiment, the present disclosure provides an engineered cell (compound CAR, ncCAR) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stimulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group, irrespective of order are CD19, BCMA, CD20, CD7 and Claudin 18.2. In further embodiment, the antigen recognition domain could VHH or scFv.

[0023] In another embodiment, the present disclosure provides an engineered cell (compound CAR, ncCAR) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stimulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group, irrespective of order are CD 19, CD7, CD20 and BCMA; at least one enhancer; wherein a high efficiency cleavage site is disposed between the chimeric antigen receptor polypeptide and enhancer. A high efficiency cleavage sites include, but not limited to, P2A, T2A, F2A and E2A. In a further embodiment, the cells comprising the enhancer are expressed in a single open reading frame (ORF) with a CAR under the control of a strong promoter.

[0024] BRIEF DESCRIPTION OF DRAWINGS

[0025] Figures 1A-1C. Overview of the nanobody (VHH antibody) discovery workflow. (1A) Alpacas were immunized over an 8-week period and then peripheral blood lymphocyte mRNA was isolated and processed into a VHH gene library. This library was transformed into phage- competent bacteria to generate a bacteriophage library, which was panned against BCMA to enrich for binding clones. High quality hits were identified by high-throughput enzyme-linked immunosorbent assays (ELISA) of individual VHH clones. VHH candidates with binding greater than 2-fold above average background were picked and sent for sanger sequencing to identify unique sequences. This strategy was also used to isolate other VHH antibodies. (IB) The camelid BCMA VHH antibody (BC4) was cloned into a pTT5-hIgGl Fc (LALA) expression vector and expressed in HEK293 cells. The protein was then purified, and purity of the purified protein was determined by SEC-HPLC. (1C) The camelid BCMA VHH antibody (BC5) was cloned into a pTT5-h!gGl Fc (LALA) expression vector and expressed in HEK293 cells. The protein was then purified, and purity of the purified protein was determined by SEC-HPLC.

[0026] Figures 2A-2C. (2A) ELISA was carried out to measure the binding affinity of the purified VHH BCMA antibodies, BC4 and BC5 to a purified human BCMA protein. The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. BCMA positive control and an isotype negative control are used as controls. (2B) ELISA was carried out to measure the binding affinity of the purified VHH BCMA antibodies to a purified human TACI protein. TACI is a protein expressed on the surface of B-cells. The binding affinity' is measured as the OD450 against the increasing titrated concentration of Ig antibody. BCMA positive control and an isotype negative control are used as controls. The camelid BC4 and BC5 nanobodies do not bind TACT. (2C) ELISA was carried out to measure the binding affinity' of the purified camelid BCMA antibodies to a purified human BAFFR protein. BAFFR is a protein expressed on the surface of B cells. The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. BCMA positive control and an isotype negative control are used as controls. The camelid BC4 and BC5 nanobodies do not bind BAFFR.

[0027] Figure 3. FACS was carried out to measure the binding affinity of the purified VHH BCMA antibodies of BC4 and BC5 to CHO KI cells expressing cell surface BCMA. The binding affinity is measured as the MFI against the increasing titrated concentration of Ig antibody. BCMA positive control and an isotype negative control are used as controls.

[0028] Figures 4A-4B. (4A) The camelid CD 19 VHH antibody (BS040-2) was cloned into a pTT5-hIgGl Fc (LAL A) expression vector and expressed in HEK293 cells. The protein was then purified, and purity of the purified protein was determined by SEC-HPLC. (4B) The camelid CD19 VHH antibody (BS040-10) was cloned into a pTT5-hIgGl Fc (LALA) expression vector and expressed in HEK293 cells. The protein was then purified, and purity of the purified protein was determined by SEC-HPLC. The strategy of cloning and isolation of both camelid CD19VHH antibody sequences for BS040-2 and BS040-10 is the same as described in the Figure 1A.

[0029] Figures 5A-5B. (5A) ELISA was carried out to measure the binding affinity of the purified camelid (VHH) CD 19 antibody (BS040-2) to a purified human CD 19 His-tag protein (human CD 19 protein with histidine tag). The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls. Tafasitamab is an Fc-modified monoclonal antibody that binds to CD 19, and a drug that was approved by the US FDA. (5B) ELISA was carried out to measure the binding affinity of the purified camelid CD 19 antibody (BS040-10) to a purified human CD 19 His-tag protein (human CD 19 protein with histidine tag). The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls. Tafasitamab is an Fc-modified monoclonal antibody that binds to CD 19, and a drug that was approved by the US FDA. Figures 6A-6B. (6A) FACS was carried out to measure the binding affinity of the purified camelid CD19 antibody (BS040-2) to Raji cells expressing CD19. The binding affinity is measured as a dot plot of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and iso type are used as controls. (6B) FACS was carried out to measure the binding affinity of the purified camelid CD19 antibody (BS040-10) to Raji cells expressing CD19. The binding affinity is measured as a dot plot of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0030] Figures 7A-7B. (7 A) FACS was carried out to measure the binding affinity of the purified camelid CD19 antibody (BS040-2) to HEK293 cells that express CD19. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls. (7B) FACS was carried out to measure the binding affinity of the purified camelid CD19 antibody (BS040-10) to HEK293 cells that express CD19. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0031] Figures 8A-8B. (8A) FACS was carried out to measure the binding affinity of the purified camelid CD 19 antibody (BS040-2) to Nalm6-Luc2 cells that express CD 19. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls. (8B) FACS was carried out to measure the binding affinity of the purified camelid CD19 antibody (BS040-10) to Nalm6-Luc2 cells that express CD 19. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0032] Figures 9A-9C. (9A) BLI assay was carried out to measure the molecular interactions between Tafasitamab and Human CD 19 His-tag. The interactions are measured as a curve graph of distance (nm) against time. (9B) BLI assay was carried out to measure the molecular interactions between CD 19 nanobody (BS040-2) and Human CD 19 His-tag. The interactions are measured as a curve graph of distance (nm) against time. (9C) BLI assay was carried out to measure the molecular interactions between CD 19 nanobody (BS040- 10) and Human CD 19 His-tag. The interactions are measured as a curve graph of distance (nm) against time.

[0033] Figure 10. The camelid Claudin 18.2 VHH antibody was cloned into a pTT5-hIgGl Fc (LALA) expression vector and expressed in HEK293 cells. The protein was then purified, and purity of the purified protein was determined by SEC-HPLC. The strategy of cloning and isolation of camelid Claudin 18.2 VHH antibody sequences is the same as described in the Figure 1A.

[0034] Figure 11. FACS was carried out to measure the binding affinity of the purified camelid Claudin 18.2 VVH antibody to HEK293 cells that express Claudin 18.2. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. VHH Fc and isotype are used as controls.

[0035] Figure 12. Bar graph of FACS carried out to measure the binding affinity of the purified camelid Claudin 18.2 VHH antibody to HEK293 cells that express Claudin 18.1. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of 1g antibody. VHH Fc and isotype are used as controls. The isolated Claudin 18.2 VHH antibody did not bind to HEK 293 expressing Claudin 18.1.

[0036] Figures 13A-13B. ELISA was carried out to measure the binding affinity of the purified humanized BCMA scFv antibodies to a purified Human BCMA or CD19 His-tag protein. (13A) The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. BCMA REGN5459 and isotype are used as controls. (13B) ELISA was carried out to measure the binding affinity of the purified humanized CD 19 scFv antibodies to a purified Human CD 19 His-tag protein. The binding affinity is measured as the OD450 against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0037] Figures 14A-14B. (14 A) FACS was carried out to measure the binding affinity of the purified humanized BCMA scFv antibodies to RPMI8226 cells that express BCMA. The binding affinity is measured as the MFI against the increasing titrated concentration of Ig antibody. BCMA REGN5459 and isotype are used as controls. (14B) FACS was carried out to measure the binding affinity of the purified humanized CD19 scFv antibodies to Raji cells that express CD19. The binding affinity is measured as the MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0038] Figures 15A-15B. (15A) FACS was carried out to measure the binding affinity of the purified humanized BCMA antibodies to HEK293 cells. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. BCMA REGN5459 and isotype are used as controls. (15B) FACS was carried out to measure the binding affinity of the purified humanized CD 19 antibodies to HEK293 cells. The binding affinity is measured as a bar graph of MFI against the increasing titrated concentration of Ig antibody. Tafasitamab and isotype are used as controls.

[0039] Figures 16A-16B. (16A) The combination of a CAR, (including, but not limited to, third or second generation), and sushi domain of the IL- 15 alpha receptor (called IL15sushi), is assembled on an expression vector and their expression is driven by a promoter. CAR with IL-15 / IL-15 sushi is linked with the P2A self-cleaving sequence. The IL-15 / IL-15sushi portion is composed of IL-2 signal peptide fused to IL- 15 and linked to sushi domain of IL- 15 alpha receptor via a 26-amino acid poly-proline linker. (16B) CAR and IL-15 / IL-l 5 sushi are present on the T orNK cells.

[0040] Figure 17. Comparison CD19-CAR-T vs CD19-IL-15 / IL15sushi CAR-T against REH cells expressing CD 19 over long term. Mice were injected with CD 19 positive REH tumor cells expressing (0.5x106cells / mouse) expressing luciferase on Day 1. On Day 3, IV1S was conducted to assay the appearance of REH cells. On Day 4, CD 19 CAR, and CD19-IL15 / IL15sushi CAR T- cells were injected (~7.5xl 06total cells / mouse) and on day 6 through 32, IVIS imaging was conducted to assay semi-quantitative assessment of tumor burden and subsequent tumor depletion and control. The study demonstrates that IL-15 / IL15sushi enhances the killing of targeted cells.

[0041] Figures 18A-18E. (18A) Schematic representation ofBC4-Q-VAC CAR construct. The BC4-Q- VAC nCAR consists of a leader sequence, an anti-BCMA (B-cell maturation antigen) nanobody (VHH, heavy chain-only antibody), a hinge domain, transmembrane (TM) regions, CD28 co- activation domains linked to the CD3C signaling domain, and a P2A self-cleaving sequence linked to the 1L-15 / 1L-I5sushi domain of the IL-15 alpha receptor. The IL-15 / IL-15sushi portion is composed of an IL -2 signal peptide fused to IL- 15 and linked to the sushi domain via a 26-amino acid poly-proline linker. The CAR includes two rituximab (RTX)-binding epitopes in the hinge region as a safety switch. (18B) Transduction of T cells with BC4-Q-VAC CAR. Activated T cells from the cord blood buffy coat were transduced with either mock (left) or BC4-Q-VAC CAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat- VHH antibody or goat IgG (control) at 1 :250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. The transduction efficiency showed a significant percentage of T cells expressing the BC4-Q-VAC CAR construct, with 50.93% positive cells. (18C) Whole-body bioluminescence images of NSG mice injected with BC4-Q-VAC nCAR-T or mock T cells. Images were taken on days 5, 8, 11, 14, and 18 to monitor tumor burden in the mice. ( 18D) Quantitation of total body bioluminescence. The bar graph shows the mean bioluminescence intensity of mock T or BC4-Q-VAC CAR-T injected mice at indicated days (n=5 mice per group). Percent tumor reduction in mice was measured and compared between the groups, indicating significant tumor reduction in BC4-Q-VAC CAR-T treated mice. (18E) Kaplan-Meier survival analysis curve of mice treated with BC4-Q-VAC CAR-T cells vs. control mock T cells. The Mantel-Cox log-rank test was used for statistical analysis. The results indicated significantly prolonged survival in mice treated with BC4-Q-VAC CAR-T cells compared to control mice (p-0.0031).

[0042] Figures 19A-19E: (19A) Schematic representation of BC5-Q-VAC CAR construct. The BC5-Q- VAC CAR includes a leader sequence, an anti-BCMA nanobody, a hinge domain, transmembrane regions, CD28 co-activation domains linked to the CD3C signaling domain, and an 1L-15 / 1L- 15sushi domain of the IL- 15 alpha receptor. (19B) Transduction of T cells with BC5-Q-VAC CAR. Activated T cells from the cord blood buffy coat were transduced with either mock (left) or BC5-Q-VAC CAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat- VHH antibody or goat IgG (control) at 1 :250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. The transduction efficiency showed a significant percentage of T cells expressing the BC5-Q-VAC CAR construct. (19C) Whole-body bioluminescence images of NSG mice injected with BC5-Q-VAC CAR-T or mock T cells. Images were taken on days 5, 8, 11, 14, and 18 to monitor tumor burden in the mice. (19D) Quantitation of total body bioluminescence. The bar graph shows the mean bioluminescence intensity of mock T or BC5-Q-VAC CAR-T injected mice at indicated days (n-5 mice per group). Percent tumor reduction in mice was measured and compared between the groups, indicating significant tumor reduction in BC5-Q-VAC CAR-T treated mice. ( 19E) Kaplan-Meier survival analysis curve of mice treated with BC5-Q-VAC CAR-T cells vs. control mock T cells. The Mantel-Cox log-rank test was used for statistical analysis. The results indicated significantly prolonged survival in mice treated with BC5-Q-VAC CAR-T cells compared to control mice (p-0.0031).

[0043] Figures 20A-20C: (20A) Schematic representation of the AB 19-N2-28-15R nCAR construct. This nanobody CAR construct comprises a leader sequence, an anti-CD 19 nanobody (VHH, heavy chain-only antibody), a hinge domain, transmembrane (TM) regions. CD28 co-activation domains linked to the CD3ζ signaling domain, and a T2A self-cleaving sequence to the IL-15 / IL-15sushi domain of the IL-15 alpha receptor. The IL-15 / IL-15sushi portion is composed of an IL-2 signal peptide fused to IL- 15 and linked to the sushi domain via a 26-amino acid poly-proline linker. (20B) Transduction of T cells with AB19-N2-28-15R nCAR. Activated T cells from cord blood buffy coat were transduced with either mock (left) or AB19-N2-28-15R nCAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat- VHH antibody or goat IgG (control) at 1 :250 for 30 minutes. Cells were washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. Transduction efficiency shows a significant percentage of T cells expressing the AB19-N2-28-15RnCAR construct. (20C) Flow cytometry analysis of mock T-cells and AB19-N2-28-15R nCAR T-cells against REH tumor target cells at 0.1:1, 0.5:1, and 1:1 E:T ratios. Cells were harvested and stained with CD19 and CD3 antibodies at 1:250 for 30 minutes, washed, suspended in 2% formalin, and analyzed by flow cytometry. The results show effective lysis of REH tumor cells by AB 19-N2-28-15R nCAR T-cells, demonstrating targeted cytotoxicity against CD19-expressing tumor cell lines. Figures 21A-21C: (21A) Schematic representation of the AB19-N2-BB nCAR construct. The nanobody CAR construct comprises a leader sequence, an anti-CD19 nanobody (VHH, heavy chain-only antibody), a hinge domain, transmembrane (TM) regions, and 4- IBB co-activation domains linked to the CD3q signaling domain. (21B) Transduction of T cells with AB19-N2-BB nCAR. Activated T cells from cord blood buffy coat were transduced with either mock (left) or AB19-N2-BB nCAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat-VHH antibody or goat IgG (control) at 1 :250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. The transduction efficiency showed a significant percentage of T cells expressing the AB 19-N2-BB nCAR construct, with 84.66% positive cells. (21C) Flow cytometry analysis of mock T-cells and AB19- N2-BB nCAR T-cells against REH tumor target cells at 0.1: 1, 0.5:1, and 1:1 E:T ratios. Cells were harvested and stained with CD 19 and CD3 antibodies at 1 :250 for 30 minutes, washed, suspended in 2% formalin, and analyzed by flow cytometry. AB19-N2-BB nCAR T-cells demonstrated targeted lysis of tumor cell lines, with effective cytotoxicity observed at all tested ratios.

[0044] Figures 22A-22F: (22 A) Schematic representation of BC4-AB19-N2-15R ncCAR construct. The nanobody compound (nc) CAR construct comprises a leader sequence, an anti-CD269 (BCMA, B-cell maturation antigen) or CD 19 nanobody (VHH, heavy chain-only antibody), a hinge domain, transmembrane (TM) regions, and 4- IBB or CD28 co-activation domains linked to the CD3ζ signaling domain. The construct includes a promoter driving the expression of two modular units of nCAR, linked by a P2A peptide, followed by the 1L-15 / IL- 15 sushi domain linked by the T2A cleaving sequence. Upon cleavage of the P2A, the BCMA and CD 19 nCARs split and engage targets expressing BCMA and CD19. (22B) Transduction of T cells with BC4-AB19-N2-15R ncCAR. Activated T cells from the cord blood buffy coat were transduced with either mock (left) or BC4-AB19-N2-15R ncCAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat-VHH antibody or goat IgG (control) at 1:250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. The transduction efficiency showed significant percentages of T cells expressing the BC4-AB19- N2-15R ncCAR construct, with 69.24% positive cells. (22C) Flow cytometry analysis of mock T- cells and BC4-AB19-N2-15R ncCAR T-cells against MM.ls tumor target cells at 0.5: 1, 1:1, and 2:1 E:T ratios. Cells were harvested and stained with BCMA antibody (MM. Is) and CD3 antibody at 1 :250 for 30 minutes. Cells were then washed, suspended in 2% formalin, and analyzed by flow cytometry. BC4-AB19-N2-15R ncCAR T-cells demonstrated targeted lysis of tumor cell lines, with effective cytotoxicity observed at all tested ratios. (22D) Flow cytometry analysis of mock T- cells and BC4-AB19-N2-15R ncCAR T-cells against REH tumor target cells at 0.1:1, 0.5:1, and 1 :1 E:T ratios. Cells were harvested and stained with CD 19 (REH) and CD3 antibody at 1:250 for 30 minutes. Cells were then washed, suspended in 2% formalin, and analyzed by flow cytometry. BC4-AB 19-N2-15R ncCAR T-cells demonstrated targeted lysis of tumor cell lines, with effective cytotoxicity observed at all tested ratios. (22E) Whole-body bioluminescence images ofNSG mice injected with BC4-AB19-N2-15R ncCAR-T or mock T cells. NSG mice were sublethally irradiated and intravenously (tail vein) injected the following day with luciferase (Luc)-expressing REH cells to induce measurable tumor formation. After 6 days, the mice were intravenously injected with one course of 10 x 10Λ6 CAR-T cells or vector control T cells. On days 5, 8, 11, 14, and 18, mice were injected subcutaneously with D-Luciferin and subjected to IVIS imaging. (22F) Whole-body bioluminescence images of NSG mice injected with BC4-AB19-N2-15R ncCAR-T or mock T cells. NSG mice were sublethally irradiated and intravenously injected with luciferase (Luc)-expressing U937-BCMA cells to induce measurable tumor formation. After 6 days, the mice were treated with a course of 10 x 10Λ6 BC4-AB19-N2-15R ncCAR T-cells or mock T cells. Bioluminescence imaging was performed on days 5, 8, 11, 14, and 18 to monitor tumor burden. The images show significant tumor reduction in the BC4-AB19-N2-15R ncCAR T-treated group compared to the control group at all time points, as indicated by the reduced bioluminescence signal in treated mice.

[0045] Figures 23A-23B: Schematic Representation of 19N10-19N2-BB ntCAR Construct

[0046] (23 A) The 19N10-19N2-BB ntCAR construct is illustrated, comprising tandem anti-CD19 N10 and N2 nanobodies connected by a linker, a hinge domain, transmembrane (TM) regions, 4- IBB co-activation domains linked to the CD3C signaling domain. (23B) Superior Anti-tumor Activity of 19N 10-19N2-BB ntCAR T Cells compared to AB 19-N2-BB and 19N 10-BB nCAR T Cells against REH Tumor Cells. Flow cytometry analysis of the tumor-killing activity of different CAR T cell constructs co-cultured with REH tumor cells. The constructs tested include Mock, N2, N10, and N2N10 tan CAR T cells at effector-to-target (E:T) ratios of 0.1:1, 0.5: 1, and 1: 1. The co- culture was incubated for 6 hours before cells were harvested, stained with CD 19 (REH) and CD3 antibodies, fixed in 2% formalin, and analyzed by flow cytometry.

[0047] Figures 24A-24B: Schematic Representation of 19N10-19N2-28-15R ntCAR Construct (24A) The 19N10-19N2-28-15R ntCAR construct is illustrated, featuring tandem anti-CD19 N10 and N2 nanobodies connected by a linker, a hinge domain, transmembrane (TM) regions, CD28 co-activation domains linked to the CD3c signaling domain, and a P2A self-cleaving sequence to the IL-15 / IL-15sushi domain of the IL- 15 alpha receptor. (24B) Cytotoxicity' of 19N 10-19N2-28- 15R ntCAR T Cells Against REH Tumor Cells. Flow cytometry' analysis of mock-transduced T cells and 19N10-19N2-28-15R ntCAR T cells co-cultured with REH tumor target cells at 0.1 : 1 , 0.5: 1, and 1 :1 E:T ratios. Cells were harvested, stained with CD19 and CD3 antibodies at 1:250 for 30 minutes, fixed in 2% formalin, and analyzed by flow cytometry. The plots show the targeted lysis of tumor cells by 19N10-19N2-28-15R ntCAR T cells.

[0048] Figures 25A-25D: (25A) Schematic representation of BC4-AB19-N2-BB ntCAR construct. The BCMA-CD19 nanobody tandem (nt) CAR construct comprises a leader sequence, tandem anti- CD269 (BCMA, B-cell maturation antigen) and anti-CD19 nanobodies (VHH, heavy chain only antibody) linked by a linker, a hinge domain, transmembrane (TM) regions, 4- IBB co-activation domains linked to the CD3C signaling domain. (25B) Transduction of T cells with BC4-AB 19-N2- BB ntCAR. Activated T cells from cord blood buffy coat were transduced with either mock (left) or BC4-AB 19-N2-BB ntCAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested and stained with goat- VHH antibody or goat IgG (control) at 1:250 for 30 minutes. Cells were washed and stained with streptavidin-PE conjugate at 1:500, washed, suspended in 2% formalin, and analyzed by flow cytometry. The transduction efficiency showed a significant percentage of T cells expressing the BC4-AB19-N2-BB ntCAR construct, with 76.38% positive cells. (25C) Flow cytometry analysis of mock T-cells and BC4- AB19-N2-BB ntCAR T-cells against MMl .s and REH tumor target cells at 0.5:1, 1 :1, and 2: 1 E:T ratios. Cells were harvested and stained with BCMA antibody (MM1.s) and CD3 antibody at 1 :250 for 30 minutes. Cells were then washed, suspended in 2% formalin, and analyzed by flow cytometry. BC4-AB19-N2-BB ntCAR T-cells demonstrated targeted lysis of tumor cell lines, with effective cytotoxicity observed at all tested ratios. (25D) Flow cytometry analysis of mock T-cells and BC4-AB19-N2-BB ntCAR T-cells against U937-BCMA and REH tumor target cells at 0.1 :1, 0.5: 1, and 1 :1 E:T ratios. Cells were harvested and stained with CD19 (REH) and CD3 antibody at 1 :250 for 30 minutes. Cells were then washed, suspended in 2% formalin, and analyzed by flow cytometry. BC4-AB19-N2-BB ntCAR T-cells demonstrated targeted lysis of tumor cell lines, with effective cytotoxicity observed at all tested ratios.

[0049] Figures 26A-26B: Schematic Representation of 19N10-28 VAC nCAR Construct

[0050] (26A) The 19N10-28 nCAR construct is illustrated, comprising a leader sequence, an anti-CD19 nanobody (VHH, heavy chain only antibody), a hinge domain, transmembrane (TM) regions, CD28 co-activation domains linked to the CD3(j signaling domain, and a P2A self-cleaving sequence to the IL-15 / IL-15sushi domain of the IL- 15 alpha receptor. (26B) Cytotoxicity of 19N10-28 VAC nCAR T Cells Against REH Tumor Cells. Activated T cells from cord blood buffy coat were transduced with either mock or 19N 10-28 VAC nCAR viral supernatant from transfected HEK-293FT cells Flow cytometry analysis of mock-transduced T cells and 19N10-28 VAC nCAR T cells co-cultured with REH tumor target cells at 0.1:1, 0.5:1, and 1 :1 E:T ratios. Cells were harvested, stained with CD19 and CD3 antibodies at 1 :250 for 30 minutes, fixed in 2% formalin, and analyzed by flow cytometry. The plots show the targeted lysis of tumor cells by 19N 10-28 VAC nCAR T cells.

[0051] Figures 27A-27D: Schematic Representation of AB19-N10-BB nCAR Construct

[0052] (27A) The AB19-N10-BB nCAR construct is illustrated, comprising a leader sequence, an anti- CD 19 nanobody (VHH, heavy chain only antibody), a hinge domain, transmembrane (TM) regions, and 4- IBB co-activation domains linked to the CD3ζ signaling domain. (27B) Transduction of T Cells with AB19-N10-BB nCAR. Flow cytometry analysis of T cells transduced with AB19-N10-BB nCAR. Activated T cells from cord blood buffy coat were transduced with either mock (left) or AB19-N 10-BB nCAR (right) viral supernatant from transfected HEK-293FT cells. After 7 days of incubation, cells were harvested, stained with goat anti-VHH antibody or goat IgG (control) at 1:250 for 30 minutes, followed by staining with streptavidin-PE conjugate at 1 :500, fixed in 2% formalin, and analyzed by flow cytometry. The flow cytometry plots show the percentage of CAR-positive cells. (27C) Cytotoxicity of AB 19-N10-BB nCAR T Cells Against REH Tumor Cells. Flow cytometry analysis of mock-transduced T cells and AB19-N10-BB nCAR T cells co-cultured with REH tumor target cells at 0.1:1, 0.5:1, and 1 :1 E:T ratios. Cells were harvested, stained with CD19 and CD3 antibodies at 1 :250 for 30 minutes, fixed in 2% formalin, and analyzed by flow cytometry. The plots show the targeted lysis of tumor cells by AB19-N10- BB nCAR T cells. (27D) Whole-body bioluminescence images of NSG mice injected with AB19- N10-BB nCAR T cells or mock T cells. NSG mice were sublethally irradiated and intravenously injected with luciferase (Luc )-expressing REH cells to induce measurable tumor formation. After 6 days, the mice were treated with a course of 10 x 10Λ6 AB19-N10-BB nCAR T-cells or mock T cells. Bioluminescence imaging was performed on days 4, 7, 11, 14, 18, and 33 to monitor tumor burden. The images show significant tumor reduction in the AB 19-N10-BB nCAR T-treated group compared to the control group at all time points, as indicated by the reduced bioluminescence signal in treated mice.

[0053] Figures 28A-28C: Characterization and Cytotoxicity of GI CAR-NK Cells

[0054] (28A) Schematic representation of the GI CAR construct. The GI CAR construct features an anti- Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a 4- IBB (BB) co-stimulatory domain, and a CD3C signaling domain. (28B) Flow cytometry analysis of CAR expression in transduced NK cells. NK cells from the cord blood buffy coat were transduced with either mock (left) or GI CAR (right) viral supernatant. After five days of incubation, cells were harvested and stained with a goat anti- VHH antibody or goat IgG (control) at 1:250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1 :500. Cells were then fixed in 2% formalin and analyzed by flow cytometry. The plots indicate the percentage of CAR-positive cells, demonstrating successful transduction and expression of the GI CAR construct in NK cells. (28C) Cytotoxicity assay of GI CAR-NK cells. Flow cytometry analysis of mock-transduced NK cells and GI CAR-NK cells co-cultured with SNU-601 target cells at different effector-to-target (E:T) ratios. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour and incubated with CAR-NK cells for 18 hours. Cells were then harvested, stained with anti-CD56 antibody to distinguish effector NK cells (left columns) from target SNU-601 cells (right upper columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by CAR-NK cells, demonstrating their cytotoxic efficacy.

[0055] Figures 29A-29C. Characterization and Cytotoxicity of GIV CAR-NK Cells

[0056] (29 A) Schematic representation of the GIV CAR construct. The GIV CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a 4-1BB (BB) co-stimulatory domain, a CD3c signaling domain, and a T2A self-cleaving sequence linked to the IL-15 / IL-15sushi cytokine module. (29B) Flow cytometry analysis of CAR expression in transduced NK cells. NK cells isolated from the cord blood buffy coat were transduced with either mock (left) or GIV CAR (right) viral supernatant. After five days of incubation, cells were harvested, stained with a goat anti-VHH antibody or goat IgG (control) at 1:250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1:500. Cells were then fixed in 2% formalin and analyzed by flow cytometry. The plots display the percentage of CAR-positive cells, confirming successful transduction and expression of the GIV CAR construct in NK cells. (29C) Cytotoxicity assay of GIV CAR-NK cells. Flow cytometry analysis of mock -transduced NK cells and GIV CAR-NK cells co-cultured with SNU-601 target cells at different effector-to-target (E:T) ratios. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells (lower right columns) from target SNU-601 cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by GIV CAR-NK cells, demonstrating their cytotoxic activity.

[0057] Figures 30A-30C. Characterization and Cytotoxicity of GI18 CAR-NK Cells

[0058] (30A) Schematic representation of the Gil 8 CAR construct. The GI18 CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a CD28 (28) co-stimulatory domain, a CD3C signaling domain, and a P2A self-cleaving sequence linked to the IL- 18m cytokine module. (30B) Flow cytometry analysis of CAR expression in transduced NK cells. NK cells isolated from the cord blood buffy coat were transduced w'ith either mock (left) or Gil 8 CAR (right) viral supernatant. After five days of incubation, cells were harvested, stained with a goat anti-VHH antibody or goat IgG (control) at 1 :250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1:500. Cells were then fixed in 2% formalin and analyzed by flow cytometry. The plots display the percentage of CAR-positive cells, confirming successful transduction and expression of the GI 18 CAR construct in NK cells. (30C) Cytotoxicity assay of GI18 CAR-NK cells. Flow cytometry analysis of mock-transduced NK cells and GI18 CAR-NK cells co-cultured with SNU-601 target cells at different effector-to-target (E:T) ratios. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells (lower right columns) from target SNU-601 cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by GI18 CAR-NK cells, demonstrating their potent cytotoxic activity.

[0059] Figures 31A-31C. Characterization and Cytotoxicity of GIV18 CAR-NK Celis

[0060] (31 A) Schematic representation of the GIVI 8 CAR construct. The GIVI 8 CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a CD28 (28) co-stimulatory domain, a CD3(^ signaling domain, and self-cleaving sequences (T2A and P2A) enabling the co-expression of IL-15 / IL-15sushi and IL-18m cytokine modules (matured form of IL-18). The IL-18m module encodes a 193-amino acid precursor synthesized without a signal peptide. Variations in co- stimulatory domains and cytokine modules are illustrated to highlight construct diversity. (3 IB) Flow cytometry analysis of CAR expression in transduced NK cells. NK cells isolated from the cord blood buffy coat were transduced with either mock (left) or GIVI 8 CAR (right) viral supernatant. After five days of incubation, cells were harvested, stained with a goat anti-VHH antibody or goat IgG (control) at 1:250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1:500. Cells were then fixed in 2% formalin and analyzed by flow cytometry'. The plots display the percentage of CAR-positive cells, confirming successful transduction and expression of the GIVI 8 CAR construct in NK cells. (31C) Cytotoxicity assay of GIVI 8 CAR-NK cells. Flow cytometry analysis of mock-transduced NK cells and GIVI 8 CAR-NK cells co-cultured with SNU-601 target cells at different effector-to- target (E:T) ratios. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells (lower right columns) from target SNU-601 cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by GIVI 8 CAR-NK cells, demonstrating their potent cytotoxic activity.

[0061] Figures 32A-32D. IL-15 and IL-18 Secretion in CAR-Transduced NK Cells and their functions

[0062] (32A) IL- 15 secretion by CAR-NK cells. CAR-NK cells (0.5 x 106cells / well) were cultured without exogenous IL- 15 or IL- 18 for four days. Culture supernatants were collected, and IL- 15 secretion levels were measured using ELISA. The bar graph shows IL-15 production in different CAR-NK cell groups, with GIV CAR-NK cells exhibiting the highest IL- 15 secretion, followed by GIVI 8 CAR-NK cells, while GI and GI18 CAR-NK cells showed no IL-15 production. (32B) IL- 18 secretion by CAR-NK cells. The culture supernatants from the same CAR-NK cell groups were also analyzed for IL-18 secretion using ELISA. The bar graph demonstrates IL- 18 production, with GI18 CAR-NK cells displaying the highest IL- 18 secretion, while GIVI 8 CAR- NK cells showed significantly lower IL- 18 levels. (32C) Fold expansion of CAR-NK cells over time. NK cells transduced with GI, GIV, GI18, or GIVI 8 CAR viral supernatants were cultured with IL-15 (10 ng / 'rnL) and / or IL-18 (10 ng / mL) for seven days. After this initial culture period, cells were maintained without IL- 15 and IL- 18 until day 15. Cell proliferation was assessed by counting viable cells using trypan blue staining on days 7, 11, and 15. The bar graph illustrates the fold expansion of NK cells at each time point, highlighting differences in proliferative capacity among the different CAR constructs. (32D) NSG mice were sub lethally irradiated and intravenously injected with luciferase (Luc)-expressing SNU-601 cells to induce measurable tumor formation. After 6 days, the mice were treated with a course of 5 x 10Λ6 CAR NK cells or mock NK cells. Bioluminescence imaging was performed on days 2, 13, 16, 30, and 42 to monitor tumor burden. Tumor burden, represented as total bioluminescence flux, was measured over time on days 2, 13, 16, 30, and 42 (left panel). The percentage of tumor regression observed in each group on days 16, 30, and 42. The results show significantly reduced tumor burden in mice treated with GIVI 8-CAR NK cells compared to mock and GIV-CAR NK-treated groups, highlighting the enhanced anti-tumor efficacy of GIVI 8-CAR NK cells. Figures 33A-33F. Characterization and Cytotoxicity of N399-20h- 19N2N 10-VAC CAR T Cells

[0063] (33A) Schematic Representation of N399-20h-19N2N10-VAC CAR Construct. The illustrated construct includes a leader sequence followed by a humanized (h) anti-CD20 single-chain variable fragment (scFv) and a tandem CD19N2 and N10 nanobodies (VHH1-VHH2, heavy-chain-only antibody). The tandem CD19N2 and N10 CAR unit comprises tandem anti-CD19 N2 and N10 nanobodies connected by a linker. These antigen-recognition domains are linked to hinge regions, transmembrane (TM) domains, and co-stimulatory domains (4- IBB or CD28) fused to a CD32 intracellular signaling domain. The construct employs a promoter driving the expression of two modular CAR units linked by a P2A self-cleaving peptide. An 1L-15 / IL-I5sushi domain (VAC) is incorporated downstream, linked via a T2A peptide sequence, allowing independent expression of both CARs and the cytokine. Upon P2A cleavage, the two CAR constructs independently recognize and engage CD20- and CD19-expressing target cells. (33B) and (33C) Transduction Efficiency of N399-20h-19N2N 10-VAC CAR in T Cells. Human cord blood-derived T cells were activated and transduced with N399-20h-19N2N10-VAC CAR-encoding retroviral particles. Following five days of culture, transduction efficiency was evaluated via flow cytometry. Cells were stained with anti-mouse F(ab')2 to detect CD20h CAR and goat anti-VHH antibody for CD19N2N10 at 1:250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1:500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. Representative dot plots show mock-transduced T cells, CD20h CAR-positive T cells (34.51% expression), and CD19N2N10 CAR-positive T cells (69.89% expression). (33D) IL-15 Secretion by N399-2011-19N2N 10-VAC CAR T Cells. CAR T cells (0.5 x 10Λ6 cells / well) were cultured for three days, and IL- 15 secretion was measured by ELISA. The results show elevated IL- 15 levels in the culture supernatant of N399-20h-19N2N10-VAC CAR T cells, while no detectable IL-15 was observed in mock-transduced T cells. (33E) and (33F) Cytotoxicity Assay of N399-20h- 19N2N10-VAC CAR T Cells. Flow cytometry analysis of mock-transduced T cells and N399- 20h-19N2N 10-VAC CAR T cells co-cultured with different target cells at different effector-to- target (E:T) ratios. IC562-CD20xp (CD20 overexpressing cell line) and K562-CD19xp (CD19 overexpressing cell line) were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR T cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD3 antibody to distinguish effector T cells (lower right columns) from target K562-CD20xp and K562-CD19xp cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by N399-2011-19N2N10-VAC CAR T cells.

[0064] Figures 34A-33F. Characterization N409-20h-19N10-VAC CAR T Cells

[0065] (34A) Schematic Representation of N409-20h-19N10-VAC CAR Construct. The illustrated construct includes a leader sequence followed by a humanized (h) anti-CD20 single-chain variable fragment (scFv) and a CD19N10 nanobody (VHH, hcavy-chain-only antibody) against CD19. These antigen-recognition domains are linked to hinge regions, transmembrane (TM) domains, and co-stimulatory domains (4-1BB or CD28) fused to a CD32 intracellular signaling domain. The construct employs a promoter driving the expression of two modular CAR units linked by a P2A self-cleaving peptide. An IL-15 / IL-15sushi domain (VAC) is incorporated downstream, linked via a T2A peptide sequence, allowing independent expression of both CARs and the cytokine. Upon P2A cleavage, the two CAR constructs independently recognize and engage CD20- and CD 19- expressing target cells. (34B) and (34C) Transduction Efficiency of N401-20h-19N10-VAC CAR in T Cells. Human cord blood-derived T cells were activated and transduced with N401-20h- 19N10-VAC CAR-encoding retroviral particles. Following five days of culture, transduction efficiency was evaluated via flow cytometry. Cells were stained with anti-mouse F(ab')2 to detect CD20h CAR and goat anti-VHH antibody for CD19N10 at 1:250 for 30 minutes. The cells were then washed, stained with streptavidin- PE conjugate at 1:500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. Representative dot plots show mock-transduced T cells, CD20h CAR-positive T cells (29.00% expression), and CD19N10 CAR-positive T cells (65.71% expression). (34D) IL- 15 Secretion by N401-20h-19N10-VAC CAR T Cells. CAR T cells (0.5x10Λ6 cells / well) were cultured for three days, and IL- 15 secretion was measured by ELISA. The results show' elevated IL- 15 levels in the culture supernatant of N401-20h-19N10-VAC CAR T cells, while no detectable IL- 15 was observed in mock-transduced T cells. (34E) and (34F) Cytotoxicity Assay of N401-2011-19N 10- VAC CAR T Cells. Flow" cytometry analysis of mock- transduced T cells and N401-2011-19N10-VAC CAR T cells co-cultured with different target cells at different effector-to-target (E:T) ratios. K562-CD20xp (CD20 overexpressing cell line) and K562-CD19xp (CD19 overexpressing cell line) were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR T cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD3 antibody to distinguish effector T cells (lower right columns) from target K562-CD20xp and K562-CDI9xp cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by N401-20h-19N10-VAC CAR T cells.

[0066] Figures 35A-35F. Characterization of N412-20h-19N2N10-18m CAR T Cells

[0067] (35A) Schematic Representation of N412-20h-19N2N10-18m CAR Construct: The illustrated construct includes a leader sequence followed by a humanized (h) anti-CD20 single-chain variable fragment (scFv) and a tandem CD19N2 and N10 nanobody (VHH, heavy-chain-only antibody). The tandem CD19N2 and N10 CAR unit comprises tandem anti-CD19 N2 and N10 nanobodies connected by a linker. These antigen-recognition domains are linked to hinge regions, transmembrane (TM) domains, and co-stimulatory domains (4-1BB or CD28) fused to a CDJL intracellular signaling domain. The construct employs a promoter driving the expression of two modular CAR units linked by a P2A self-cleaving peptide. An 18m (matured form of IL- 18) domain is incorporated downstream, linked via a T2A peptide sequence, allowing independent expression of both CARs and the cytokine. Upon P2A cleavage, the two CAR constructs independently recognize and engage CD20- and CD19-expressing target cells. The 18m module encodes a 157-amino acid cytokine lacking a signal peptide. (35B) and (35C) Transduction Efficiency of N412-2011-19N2N10-18m CAR in T Cells: Human cord blood-derived T cells were activated and transduced with N412-20h-19N2N10-18m CAR-encoding retroviral particles. Following five days of culture, transduction efficiency was evaluated via flow cytometry. Cells were stained with anti-mouse F(ab’)2 to detect CD20h CAR and goat anti- VHH antibody for CD19N2N10 at 1:250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1:500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. Representative dot plots show mock-transduced T cells, CD20h CAR-positive T cells (26.69% expression), and CD19N2N10 CAR-positive T cells (73.16% expression). (35D) IL- 18 Secretion by N412-20h-19N2N10-18m CAR T Cells: CAR T cells (0.5 x 106cells / well) w'ere cultured for three days, and IL- 18 secretion was measured by ELISA. The results show elevated IL- 18 levels in the culture supernatant of N412-20h-19N2N 10-18m CAR T cells, while no detectable IL-18 was observed in mock- transduced T cells. (35E) and (35F) Cytotoxicity Assay of N412-20h- 19N2N10-18m CAR T Cells. Flow cytometry analysis of mock-transduced T cells and N412-20h- 19N2N10-18m CAR T cells co-cultured with different target cells at different effector-to-target (E:T) ratios. K562-CD20xp (CD20-overexpressing cell line) and K562-CD19xp (CD19- overexpressing cell line) were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR T cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD3 antibody to distinguish effector T cells (lower right columns) from target K562-CD20xp and K562-CD19xp cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by N412-20h-19N2N10-18m CAR T cells. The results demonstrated effective tumor cell lysis, with 21% and 67% killing of K562-CD20xp cells at 1:1 and 2: 1 ratios, respectively, and 15% and 88% lysis of K562-CD19xp cells.

[0068] Figures 36A-36E. Characterization of N413-20h-19N10N2 CAR T Cells

[0069] (36A) Schematic Representation of N413-20h-19N10N2 CAR Construct: The illustrated construct includes a leader sequence followed by a humanized (h) anti-CD20 single-chain variable fragment (scFv) and a tandem CD19N10 and N2 nanobody (VHH1-VHH2, heavy-chain-only antibody) against CD19. The tandem CD19N10 and N2 CAR unit comprises two anti-CD19 nanobodies (N10 and N2) connected by a linker. These antigen-recognition domains are linked to hinge regions, transmembrane (TM) domains, and co-stimulatory domains (4- IBB or CD28) fused to a CD3q intracellular signaling domain. The construct employs a promoter driving the expression of two modular CAR units linked by a P2A self-cleaving peptide. Upon P2A cleavage, the two CAR constructs independently recognize and engage CD20- and CD19-expressing target cells.

[0070] (36B) and (36C) Transduction Efficiency of N413-20h-19N10N2 CAR in T Cells: Human cord blood-derived T cells were activated and transduced with N413-20h-19N10N2 CAR-encoding retroviral particles. Following five days of culture, transduction efficiency was evaluated via flow cytometry. Cells were stained with anti-mouse F(ab')2 to detect CD20h CAR and goat anti-VHH antibody for CD19N10N2 at 1:250 for 30 minutes. The cells were then washed, stained with streptavidin-PE conjugate at 1 :500, washed again, suspended in 2% formalin, and analyzed by flow cytometry. Representative dot plots show mock-transduced T cells, CD20h CAR-positive T cells ( 10.02% expression), and CD19N10N2 CAR-positive T cells (49.13% expression). (36D) and (36E) Cytotoxicity Assay of N413-2011-19N10N2 CAR T Cells: Flow cytometry analysis of mock-transduced T cells and N413-20h-19N10N2 CAR T cells co-cultured with different target cells at an effector-to-target (E:T) ratio of 2: 1. K562-CD20xp (CD20-overexpressing cell line) and K562-CD19xp (CD19-overexpressing cell line) were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR T cells for 18 hours. Following incubation, cells were harvested, stained with anti-CD3 antibody to distinguish effector T cells (lower right columns) from target K562-CD20xp and K562-CD19xp cells (upper left columns), and analyzed by flow cytometry. The plots illustrate the targeted lysis of tumor cells by N413-20h-19N10N2 CAR T cells. The cytotoxicity assay demonstrated that N413-20h-19N10N2 CAR T cells efficiently eliminated CD20- and CD 19-expressing target cells. When co-cultured with K562-CD20xp cells at an effector-to-target (E:T) ratio of 2:l, N413-2011-19N10N2 CAR T cells exhibited 31% target cell killing. Similarly, when N413-20h-19N10N2 CAR T cells were co-cultured with K562- CD19xp cells, a target cell killing of 88% was observed at a 2:1 E:T ratio.

[0071] Figures 37A-36E. Screening and isolation of CLL1 -specific nanobody (VHH) antibody candidates

[0072] (37A) Expression and Purification of CLL1 -Specific Nanobody (VHH) Candidates

[0073] Left side, SDS-PAGE Analysis of Purified CLL1 VHH Candidates. Recombinant LL50, LL56, and LL88 nanobody proteins were expressed in an E. coli expression system, purified using affinity chromatography, and analyzed via SDS-PAGE under reducing conditions. The gel image confirms the presence of high-purity recombinant VHH proteins. Protein purity was assessed by Coomassie Brilliant Blue staining, indicating efficient nanobody purification. Right side, Size- Exclusion Chromatography (SEC)-HPLC Analysis of Purified CLL1 VHH Candidates. The structural integrity and monodispersity of LL50 (B), LL56 (C), and LL88 (D) were evaluated using size-exclusion chromatography (SEC). Chromatograms demonstrate sharp, single peaks, confirming that each VHH candidate exists predominantly as a monomer in solution, with minimal aggregation or degradation.

[0074] (37B) cell-based binding assay of CLL1 -specific nanobody (VHH) candidates- The binding of CEL 1 -specific nanobody (VHH) candidates, including LL50, LL56, LL88, and additional VHH variants, was assessed using HEK293 cells overexpressing CLL 1. VHH candidates were incubated with CLL 1 -expressing HEK293 cells, followed by detection with a secondary fluorophore- conjugated antibody and analysis via flow cytometry. The binding curves demonstrate dose- dependent interaction of each nanobody with CLL1. The table on the right side summarizes the half-maximal effective concentration (EC50) values for LL50, LL56, LL88, and additional VHH candidates. LL50, LL56 and LL88 exhibited the lower EC50 values, indicating the higher binding affinity for CLL1 -expressing cells. The remaining VHH candidates showed varying degrees of binding efficiency.

[0075] (37C) ELISA-based binding assay of CLL1 -specific nanobody (VHH) antibody candidates-The binding affinity of CLL1 -specific nanobody (VHH) candidates, including LL50, LL56, LL88, and additional VHH variants, was assessed using an enzyme-linked immunosorbent assay (ELISA). The assay was conducted using recombinant human CLLl-His protein immobilized onto ELISA plates. VHH candidates were incubated with the coated CLL1 protein, followed by detection with a horseradish peroxidase (HRP)-conjugated secondary antibody to measure binding interactions. The resulting binding curves illustrate dose-dependent binding of each nanobody to CLL1. The half-maximal effective concentration (EC50) values were calculated for LL50, LL56, LL88, and additional VHH candidates, providing a quantitative measure of binding affinity.

[0076] (37D) Summary of BLI binding kinetics for CLL1 -specific nanobody (VHH) antibody candidates- The binding kinetics of CLLl-specific nanobody (VHH) candidates, including LL50, LL56, LL88, and additional VHH variants, were assessed using biolayer interferometry (BLI). The assay was conducted by immobilizing human CLLl-His protein onto biosensor surfaces, followed by exposure to nanobody candidates at varying concentrations. The association and dissociation phases were measured to determine binding parameters. The table presents the kinetic rate constants, including the association rate constant (Ka), dissociation rate constant (Kdis), and equilibrium dissociation constant (KD) for LL50, LL56, LL88, and additional VHH variants.

[0077] Figures 38A-38C. (38A) Schematic Representation of N414-LL56-33B CAR Construct. The N414-LL56-33B CAR construct consists of an anti-CLLl LL56 nanobody (VHH) and an anti- CD33 single-chain variable fragment (scFv), designed for dual targeting of CLL1- and CD33- expressing tumor cells. The construct contains two complete units of CARs. Each includes leader sequence, hinge regions, transmembrane (TM) domains, and a 4- IBB or CD28 co-stimulatory domain fused to a CD3(^ intracellular signaling domain to enhance T cell activation and cytotoxicity. Expression of the CAR construct is controlled by a viral promoter, with two modular CAR units linked by a P2A self-cleaving peptide, allowing independent expression of both CARs. (38B) Cytotoxicity Assay and Target Cell Killing Efficiency of N414-LL56-33B CAR T Cells (also called N414). The tumor-killing ability of N414-LL56-33B CAR T cells was evaluated against REH-CLLlxp (CLL1 -overexpressing) and REH-CD33xp (CD33-overexpressing) target cells. Target cells were co-cultured with N414-LL56-33B CAR T cells at effector-to-target (E:T) ratios of 2:1 and 4:1 for 18 hours. After co-culture, cells were harvested, washed, and stained with anti-CLLl or anti-CD33 antibody in combination with anti-CD3 antibody to distinguish effector cells from target cells. Flow cytometry analysis demonstrated efficient target cell killing by N414-LL56-33B CAR T cells.

[0078] Quantitative analysis of target cell killing showed that N414-LL56-33B CAR T cells exhibited 55% and 82% cytotoxicity at E:T ratios of 2: 1 and 4:1, respectively, when co-cultured with REH-CLLlxp cells. Similarly, in co-culture with REH-CD33xp target cells, N414-LL56-33B CAR T cells achieved 75% and 95% cytotoxicity at E:T ratios of 2: 1 and 4: 1 , respectively(38C). These results confinn the potent tumor-killing ability of N414-LL56-33B CAR T cells against CLL1- and CD33-positive target cells.

[0079] Figures 39A-39C. (39A). Schematic Representation ofN415-LL56-33B-18m CAR Construct (also called N415). The N415-LL56-33B-18m CAR construct consists of an anti-CLLl LL56 nanobody (VHH) and an anti-CD33 single-chain variable fragment (scFv), enabling dual targeting of CLL1- and CD33 -expressing tumor cells. The construct has two complete units of CARs. Each includes leader sequence, hinge regions, antigen recognition domain, transmembrane (TM) domains, and a 4-1BB or CD28 co-stimulatory domain fused to a CD3C intracellular signaling domain to enhance T cell activation and cytotoxicity. Additionally, the secreted IL- 18m domain is incorporated via a T2A self-cleaving peptide, allowing independent expression of CAR and IL- 18m to further enhance T cell expansion, persistence, and tumor- killing activity.

[0080] (39B) and (39C). Cytotoxicity Assay and Target Cell Killing Efficiency of N415-LL56-33B- 18m CAR T Cells. The cytotoxic potential of N415-LL56-33B-18m CAR T cells was assessed against REH-CLLlxp (CLL1 -overexpressing) (39B) and REH-CD33xp (CD33-overexpressing) target cells (39C). Target cells were co-cultured with N415-LL56-33B-18m CAR T cells at effector-to-target (E:T) ratios of 2: 1 and 4: 1 for 18 hours. Following co-culture, cells were harvested, washed, and stained with anti-CLLl or anti-CD33 antibody in combination with anti- CD3 antibody to differentiate effector cells from target cells. Flow cytometry analysis confirmed efficient target cell killing by N415-LL56-33B-18m CAR T cells.

[0081] Figures 40A-40C. (40A). Schematic Representation of N420-LL56-33B-15sushi CAR Construct (also called N420). The N420-LL56-33B-15sushi CAR construct consists of an anti-CLLl LL56 nanobody (VHH) and an anti-CD33 single-chain variable fragment (scFv), enabling dual targeting of CLL1- and CD33-expressing tumor cells. The construct bears two complete units of CARs. Each includes leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and a 4-1 BB or CD28 co-stimulatory domain fused to a CD32 intracellular signaling domain to enhance T cell activation and cytotoxicity. Additionally, the secreted IL-15 / IL-15sushi domain is incorporated via a T2A self-cleaving peptide, allowing independent expression of CAR and IL-15sushi to further enhance T cell survival, expansion, and tumor-killing activity.

[0082] (40B) and (40C). Cytotoxicity Assay and Target Cell Killing Efficiency of N420-LL56-33B-IL- 15sushi (also called N420-LL56-33B-IL-15 / IL15sushi) CAR T cells. The cytotoxic potential of N420-LL56-33B-15sushi CAR T cells was assessed against REH-CLLlxp (CLL1- overexpressing) (40B) and REH-CD33xp (CD33-overexpressing) target cells (40C). Target cells were co-cultured with N420-LL56-33B-15sushi CAR T cells at effector-to-target (E:T) ratios of 2:1 and 4:1 for 18 hours. Following co-culture, cells were harvested, washed, and stained with anti-CLLl or anti-CD33 antibody in combination with anti-CD3 antibody to differentiate effector cells from target cells. Flow cytometry analysis confirmed efficient target cell killing by N420-LL56-33B-15sushi CAR T cells.

[0083] Figures 41. Tumor Regression in N373-GI (called GI as well) CAR T-Treated Mice. To assess the in vivo anti-tumor efficacy of N373-GI CAR T cells, an NSG xenograft model was established using luciferase (Luc)-expressing SNU-601 tumor cells. NSG mice were sub lethally irradiated and injected intraperitoneally (IP) with Luc-SNU-601 tumor cells to allow for measurable tumor burden. After six days, the mice were randomized and intravenously (IV) injected with 10 x 106N373-GI CAR T cells or mock-transduced T cells (control group). Tumor progression and CAR T cell-mediated clearance were monitored using whole-body bioluminescence imaging (IVIS system) at multiple time points post-treatment.

[0084] Tumor regression was quantified as a percentage reduction in bioluminescence intensity relative to baseline measurements. The N373-GI CAR T cell-treated group demonstrated a substantial and sustained reduction in tumor burden over time compared to mock T cell-treated mice, confirming the potent in vivo efficacy of N373-GI CAR T cells in targeting Claudin 18.2- expressing tumors.

[0085] Figures 42. Tumor Regression Analysis in N374-GI-VAC CAR (Also called GI-VAC) T- Treated Mice. To evaluate the in vivo anti-tumor efficacy of N374-GI-VAC CAR T cells, an NSG xenograft model was established using luciferase (Luc (-expressing SNU-601 tumor cells. NSG mice were sub lethally irradiated and injected intraperitoneally (IP) with Luc-SNU-601 tumor cells to allow for measurable tumor burden. After six days, the mice were randomized and intravenously (IV) injected with 10 x 106N374-GI-VAC CAR T cells or mock-transduced T cells (control group). Tumor progression and CAR T cell-mediated clearance were monitored using whole-body bioluminescence imaging (IVIS system) at multiple time points post- treatment. Regression was quantified by calculating the percentage reduction in total bioluminescence intensity' relative to the initial tumor burden. N374-GI-VAC CAR T cell-treated mice exhibited a greater tumor regression percentage over time compared to the mock T cell- treated control group, demonstrating the enhanced anti-tumor efficacy of N374-GI-VAC CAR T cells in vivo.

[0086] DETAILED DESCRIPTION

[0087] The gold standard for developing CAR-T cells is fusing a part of murine monoclonal antibodies raised in mice to an intracellular T-cell activation domain. The hybrid nature of this mouse / human T cell provokes an immune response as they are recognized as “non-self leading to their depletion. The longevity of the engineered CAR-T cell within the patient’s immune system is critical to the success of the CAR-T therapy as well as to longer remission times. Therefore, this immune attack on the CAR-T cells reduces the efficacy of the CAR-T cell therapy, results in higher relapse rates and poor patient outcomes. In addition to persistence of the CAR-T cells, other contributing factors to the high relapse rates observed after traditional CAR-T cell therapy can be attributed to T-cell exhaustion and escape from CAR T-cell killing primarily due to antigen loss.

[0088] The typical CAR structure includes the fusion of a variable heavy-chain (VH) and VL domains to produce an scFv, where the VH and VL are connected by a linker. Although a small sequence of amino acids, the linker can elicit an immune response by generating neutralizing antibodies against the linker and reducing the effectiveness of the CAR-T therapy. Additionally, mispairing between multiple VHH and VLL regions can result in aggregation of the scFv on the surface of the CAR- T cell and contribute to CAR-T cell exhaustion. Additionally, the scFv structure may exhibit instability when binding to the antigen, and result in T cell exhaustion. Furthermore, derivation from a murine source results in an immune response against the CAR-T cell as the recipients immune system recognizes these cells as “non-self ’, leading to their depletion. A major contributor to the efficacy of the CAR-T therapy is the longevity' of the infused CAR-T cells within the patient, their ability to proliferate and persist, resulting in longer periods of time during which they can target cancer cells, eliminating cancers and resulting in long remission periods and increased relapse free rates. Therefore, a CAR-T cell generated utilizing a mouse-derived antibody that possesses a linker polypeptide is less than optimal and contributes to higher relapse rates, reduced potential of reinfusion after the first treatment and overall poor patient outcomes.

[0089] Taken together, these challenges pose an exceeding need for the development of modified CAR- T cell therapies to improve efficacy, increase T-cell persistence, decrease T-cell exhaustion and reduce remission rates, thereby allowing for better patient outcomes.

[0090] CAR T cell therapies have shown remarkable responses, but some patients have no responses to the CAR treatment or relapse soon after achieving responses. Downregulation or loss of targeted antigen expression is noted. One of solutions is to design the CAR construct, such as bi-epitope CAR on T cells with different antigen binding domains. Each specific to a distinct epitope expressed on the cancer cell, increasing the likelihood of recognizing and killing the tumor cell. If a tumor cell loses expression of one target antigen, the bi-epitope CAR T cell is still able to bind the other antigen, potentially preventing tumor escape. Single variable domain on a heavy chain (VHH) antibody, also referred to as nanobodies provides a solution to the deficiencies in the prior art.

[0091] To tackle immunogenicity against the linker polypeptide, a CAR-T cell possessing a BCMA and CD 19 VHH derived from the camelid family that possesses a single VHH chain, eliminating the need for a linker polypeptide is provided. The absence of the linker polypeptide results in a drastically reduced risk of immunogenicity against the engineered CAR-T cell, potentiating higher efficacy of the CAR-T cell therapy. The elimination of the linker polypeptide described herein, also eliminates the potential of aggregation, reducing exhaustion of the CAR-T cells, allowing them to kill cancer cells more effectively. Furthermore, the amino acid sequence of the camelid- derived antibody provided herein more closely resembles the human antibody when compared to a murine-derived antibody. Therefore, utilizing camelid-derived BCMA and CD 19 antibodies reduce risks of immunogenicity against the engineered CAR-T cell.

[0092] FDA approved CD 19 CAR uses the murine derived scFv FMC63 clone which has demonstrated induction of neutralizing antibodies against the CAR after infusion, impairing the potential to re- dose the patient. Additionally, the scFv structure may exhibit instability when binding to the antigen, and result in T cell exhaustion. To address such limitations, this study aimed to produce a novel CAR construct utilizing camelid-derived nanobodies that target both B and plasma cells.

[0093] The process of humanization includes converting the part of the mouse antibody fused to the T- cell into an engineered protein that more closely resembles a human protein. This prevents the immune response that would traditionally occur when using a mouse antibody. The low immunogenicity of the humanized CAR-T cell allows for the expansion of humanized CAR-T cells and longer persistence of these CAR-T cells within the patient, thereby resulting in longer cancer cell killing periods, elimination of the cancer and longer remission periods. Additionally, development of the humanized CAR-T cell results opens doors to the possibility of a second round of CAR-T cell therapy should the efficacy of the first round of therapy fall. Patients who lose CAR-T cell persistence and efficacy of the CAR-T cell therapy are then eligible for a reinjection of CAR-T cells to reestablish an effective CAR-T cell population. While reinfusion with traditional murine CAR-T cells have had poor outcomes, Phase I clinical trials utilizing humanized CD 19 CAR-T cells show promising indicators such as 100% response rate in patients with B-cell ALL treated with humanized CAR-T therapy and an 84% relapse-free survival rate. Phase I clinical trials also indicate that 64% of a cohort that received a second round of humanized CAR-T therapy not only showed increased persistence of CAR-T cells, marked by B-cell aplasia, but also a 74% 1-year relapse-free survival rate. Similarly, while reinfusion with traditional murine CAR-T cells has had poor outcomes, the herein camelid BCMA and CD 19 VHH antibodies provided a superior outcome.

[0094] Accordingly, herein we described the generation of a humanized BCMA and CD 19 CAR-T cell that was designed using machine learning, purified and validated, and finally selected based on its ability to bind the target protein in multiple different cell lines in vitro.

[0095] Taken together, the engineered CAR-T cell described herein is far more superior to the prior art BCMA-CD19 CAR-T cells used to treat B cell malignancies and autoimmune conditions that are currently the standard of care.

[0096] A BCMA VHH nanobody library was constructed by immunizing alpacas with a human BCMA antigen combined with a powerful phase display technology. This approach is more effective than that of synthetic antibody library to isolate BCMA VHH nanobodies. The isolated BCMA VHH antibodies through screenings were expressed, purified, validated, and finally selected based on their ability to bind the target protein in multiple different cells in vitro using different approaches. A similar approach was utilized to generate CD 19 VHH nanobodies.

[0097] Overview and Definitions

[0098] A traditional CAR consists of six domains, namely: (1) a leader sequence, (2) a monoclonal antibody-derived single-chain variable fragments, (3) a hinge region, (4) a transmembrane domain, (5) an intracellular co-stimulatory activation domains (i) CD28 or (ii) CD 137 (4- IBB), CD27, or other co-stimulatory domains and a (6) CD3-zeta signaling domain. CAR technology has evolved from the first-generation (no co-stimulation domains) to the second-generation (one co-stimulation domain) to the third-generation CAR (many co-stimulation domains).

[0099] In an embodiment, the present disclosure provides an engineered cell (compound CAR (“cCAR) or nanobody antibody CAR “ncCAR”) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stimulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group consisting of CD 19 and BCMA. In further embodiment, the antigen recognition domain could a VHH or scFv.

[0100] In another embodiment, the present disclosure provides an engineered cell (cCAR or ncCAR) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stimulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group consisting of CD 19 and CD7. In further embodiment, the antigen recognition domain could a VHH or scFv.

[0101] In another embodiment, the present disclosure provides an engineered cell (cCAR or ncCAR) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stimulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group consisting of BCMA and CD20. In further embodiment, the antigen recognition domain could a VHH or scFv.

[0102] In another embodiment, the present disclosure provides an engineered cell (cCAR or ncCAR) including a first chimeric antigen receptor polypeptide including a first signal peptide, first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first co-stiniulatory domain; and a second chimeric antigen receptor polypeptide including a second signal peptide, second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second co-stimulatory domain; wherein the first antigen recognition domain and second antigen recognition domain are different; and the first antigen recognition domain and second antigen domain are selected from the group consisting of CD 19 and CD20. In further embodiment, the antigen recognition domain could a VHH or scFv.

[0103] In one embodiment, a CAR can be a dual CAR which can be a bispecific tandem CAR, compound CAR, cistronic chimeric antigen receptor CAR and bispecific CAR is utilized in the methods. The structure of compound CAR (cCAR) and methods of generating cCAR are described in PCT / US2016 / 039306, PCT / US2016 / 068349, PCT / US2018 / 038529, all of which are hereby incorporated by reference in their entirety.

[0104] In further embodiment, the dual CAR can be a bispecific tandem CAR (tandem CAR) described in more detail publication (Mohanty et al, https: / / doi.org / 10.3892 / or.2019.7335, Pages: 2183-2195, ONCOLOGY REPORTS ), the contents of which are incorporated herein by reference. Bispefic tandem CAR can have two particular antigen recognition sites that are joined by a peptide linker, placed in tandem on a single intracellular domain and expressed as a single CAR on a cell surface.

[0105] Bi-epitope tandem CAR has a dual domain to bind two different parts of the antigen within one Bi-CAR. The structure of a dual domain is formed with scFvl-scFv2 or VHH1-VHH2. Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain. In a preferred embodiment, each engineered CAR unit polynucleotide has different nucleotide sequences in order to avoid homologous recombination.

[0106] In one embodiment, bi-epitope tandem CAR has a dual domain to bind two different parts of the antigen within one Bi-CAR. The structure of a dual domain is formed with two different camelid CD 19 nanobody antibodies, VHH1-VHH2 (N2-N10). Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain.

[0107] In another embodiment, the present disclosure provides an engineered polypeptide including a chimeric antigen receptor (CAR or cCAR) and at least one enhancer, wherein a high efficiency cleavage site is disposed between the chimeric antigen receptor polypeptide and enhancer. A high efficiency cleavage sites include, but not limited to, P2A, T2A, F2A and E2A. In a further embodiment, the cells comprising the enhancer are expressed in a single open reading frame (ORF) with a CAR under the control of a strong promoter. In a further embodiment, the enhancer promotes CAR transduced cell expansion, persistency as well as strengthen the immune system to prevent from infections.

[0108] In a further embodiment, an enhancer can be selected from at least one of the group including, but not limited, IL-2, IL-7, IL-12, IL-15, IL-15 / IL-15sush, IL-15 / IL-15sushi anchor, IL-15 / IL-15RA, IL- 18, IL-21, IL-21 anchor, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, cytoplasmic domain of IL-15 receptor alpha, 4-1BBL, IL-21, IL -21 anchor and TGFR beta, receptors.

[0109] In another embodiment, an enhancer can be selected from elected from at least one of the group including, but not limited IL-15 / IL15sushi, IL- 15 and IL- 18. In an embodiment, an enhancer can be IL-15 / IL15sushi or IL-18. In a further embodiment, enhancers can be IL-15 / IL15sushi and IL- 18 proving a synergistic effect when two are combined.

[0110] In another embodiment, the present disclosure provides an engineered cell having the engineered polypeptide or polynucleotide described above. In another embodiment, the present disclosure identifies several camelid BCM A nanobody antibodies (BCMA VHH).

[0111] In another embodiment, the present disclosure identifies several camelid CD 19 nanobody antibodies (CD 19 VHH).

[0112] In another embodiment, the present disclosure identifies a camelid Claudin 18.2 nanobody antibody (Claudin 18.2 VHH).

[0113] In another embodiment, the present disclosure identifies several constructs for a humanized BCMA antibody.

[0114] In another embodiment, the present disclosure identifies several constructs for a humanized CD 19 antibody.

[0115] The term "ligand" as used herein refers to a chemical entity having a capacity for binding to a target. A ligand could comprise a peptide, a nucleic acid, an antibody or fragment thereof, a nucleic acid-protein fusion, and / or any other affinity agent.

[0116] Immune Cells

[0117] Immune cells or immunomodulatory cells include, but are not limited to, T cells, macrophage, NK cells and NK T cells have been used for treatment of autoimmune disorders, autoreactive cells targeted to cells, tissues, and organs, infectious diseases and cancers. In the preferred embodiment, T-cells engineered to express CAR unit or units targeted toward disease have indicated profound specificity and cytotoxicity. In another embodiment, immune cells or immunomodulatory cells including but not limited to T-cells, macrophage, NK cells and dendritic cells are engineered to express CAR unit or units targeted toward disease have indicated profound specificity and cytotoxicity.

[0118] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMC), leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood. The potential disadvantages of using NK cells in a cellular therapy include a lack of persistency that may reduce long-term efficacy.

[0119] In one embodiment, the present disclosure comprises a method of modified NK cells with long- lived or long persistency in vivo for treating a disease. Surprisingly, it is found that NK cells co- expressing IL-15 / 'IL-15sushi or IL-15 / IL-15sushi and IL- 18 can extend survival for a long period of time.

[0120] CAR organization

[0121] A "signal peptide" includes a peptide sequence that directs the transport and localization of the peptide and any attached polypeptide within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0122] The signal peptide is a peptide of any secreted or transmembrane protein that directs the transport of the polypeptide of the disclosure to the cell membrane and cell surface and provides correct localization of the polypeptide of the present disclosure. In particular, the signal peptide of the present disclosure directs the polypeptide of the present disclosure to the cellular membrane, wherein the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is in the cytoplasmic portion, or interior of the cell.

[0123] In one embodiment, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e. is a cleavable signal peptide. In an embodiment, the signal peptide is human protein of type I, II, III, or IV. In an embodiment, the signal peptide includes an immunoglobulin heavy chain signal peptide.

[0124] The hinge sequence may be obtained including, for example, from any suitable sequence from any genus, including human or a part thereof. Such hinge regions are known in the art. In one embodiment, the hinge region includes the hinge region of a human protein including CD-8 alpha, CD28, 4-1BB, 0X40, CD3-zeta, T cell receptor a or (3 chain, a CD3 zeta chain, CD28, CD3s, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, GDI 37, ICOS, hemagglutinin (HA) of influenza vinis, glycosylphosphatidylinositol (GPI)-anchored protein, CD 154 and functional derivatives thereof, and combinations thereof. In one embodiment, the hinge region includes the CD8a hinge region.

[0125] In some embodiments, the hinge region includes one selected from, but is not limited to, immunoglobulin (e.g. IgGl, IgG2, IgG3, IgG4, and IgD).

[0126] The transmembrane domain includes a hydrophobic polypeptide that spans the cellular membrane. In particular, the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic).

[0127] The transmembrane domain may be in the form of an alpha helix or a beta barrel, or combinations thereof. The transmembrane domain may include a polytopic protein, which has many transmembrane segments, each alpha-helical, beta sheets, or combinations thereof.

[0128] The transmembrane sequence may be obtained including, for example, from any suitable sequence from any genus, including human or a part thereof. Such transmembrane regions are known in the art. In one embodiment, the transmembrane region includes the transmembrane region of a human protein including a T-cell receptor a or β chain, a CD3 zeta chain, CD28, CD3c, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, hemagglutinin (HA) of influenza virus, and functional derivatives thereof, and combinations thereof. In one embodiment, the transmembrane region includes the CD8a transmembrane region.

[0129] In an embodiment, the CAR polypeptide further includes one or more co-stimulatory domains. In an embodiment, the co-stimulatory domain is a functional domain from a protein including CD27, CD2, CD7, CD28, CD30, CD40, PD-1, CD258, 0X40, Natural killer Group 2 member C (NKG2C), Natural killer Group 2 member D (NKG2D), B7-H3, a ligand that binds to CD83, ICAM-1, LFA-I (CD1 la / CD 18), ICOS and 4- IBB (CD 137), active fragments thereof, functional derivatives thereof, and combinations thereof. Such co-stimulatory domains are known in the art.

[0130] In an embodiment, the signaling domain includes the polypeptide of a functional domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RI1IA, FcR beta (Fc Epsilon Rib), CD3 gamma. CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP 10), DNAX- activating protein 12 (DAP 12), active fragments thereof, functional derivatives thereof, and combinations thereof. Such signaling domains are known in the art.

[0131] In one embodiment, the antigen recognition domain includes fragment antigen-binding fragment (Fab). In another embodiment, the antigen recognition domain includes a single-chain variable fragment (scFv). scFV is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide. Such antigen recognition domain including a single-chain variable fragment is known in the art.

[0132] In another embodiment, the antigen recognition domain includes Camelid single domain antibody, or moieties thereof. In one embodiment, Camelid single-domain antibodies include heavy-chain antibodies found in camelids, or VHH antibody. A VHH antibody of camelid (for example camel, dromedary, llama, and alpaca) refers to a variable fragment of a camelid single-chain antibody (See Nguyen et al, 2001; Muyldermans, 2001), and also includes an isolated VHH antibody of camelid, a recombinant VHH antibody of camelid, or a synthetic VHH antibody of camelid. Such single-domain antibodies are known in the art.

[0133] Sources of Cells

[0134] The engineered cells may be obtained from peripheral blood, cord blood, bone marrow, tumor infiltrating lymphocytes, lymph node tissue, or thymus tissue. The host cells may include placental cells, embryonic stem cells, induced pluripotent stem cells, or hematopoietic stem cells. The cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. The cells may be obtained from established cell lines.

[0135] The above cells may be obtained by any known means. The cells may be autologous, syngeneic, allogeneic, or xenogeneic to the recipient of the engineered cells.

[0136] The term "autologous" refer to any material derived from the same individual to whom it is later to be re-introduced into the individual. The term "allogeneic" refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenic ally. The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0137] The term "syngeneic" refers to an extremely close genetic similarity or identity especially with respect to antigens or immunological reactions. Syngeneic systems include for example, models in which organs and cells (e.g. cancer cells and their non-cancerous counterparts) come from the same individual, and / or models in which the organs and cells come from different individual animals that are of the same inbred strain.

[0138] In certain embodiments, T and NK cells are derived from human peripheral blood mononuclear cells (PBMC), leukapheresis products (PBSC), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0139] The potential disadvantages of using NK cells as therapy include a lack of persistency that may reduce long-term efficacy.

[0140] In some embodiments, engineered cells can immune cells or non-immune cells. Non-immune cells, for instance, can be red blood cells as a carrier to cany cytokines or chemokines to the infected and cancer tissues.

[0141] In accordance with the present disclosure, red blood cells as a carrier provide a readily available cell to be engineered to contain at least one cytokine or chemokine selecting from a group of cytokines or chemokines including, but not limited to, IL- 15, IL-15 / IL-15sush, IL-15 / IL-15RA ( full length of IL-15 receptor a ), IL- 15 / IL-l 5 anchor, IL-2, IL-7, IL-12, IL-18, IL-21, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CXCL1, CXCL2, CXCL9, CXCL10, CXCL12 and CCL-21 polypeptide disclosed

[0142] In an embodiment, the engineered cells include immunoregulatory cells. Engineered immunoregulatory cells include T-cells, such as CD4 T-cells (Helper T-cells), CDS T- cells (Cytotoxic T-cells, CTLs), and memory T cells or memory stem cell T cells. In another embodiment, T-cells include Natural Killer T-cells (NK T-cells) and gamma delta (yδ) T cells.

[0143] In an embodiment, immunoregulatory cells can be derived from embryonic stem cells or induced pluripotent stem cells (IPS cells)

[0144] In an embodiment, the engineered cell includes Natural Killer cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells.

[0145] In an embodiment, the engineered cell includes macrophages.

[0146] NK cells mediate anti-tumor effects without the risk of GvHD and are short-lived relative to T- cells. Accordingly, NK cells would be exhausted shortly after destroying targeted cells, decreasing the need for an inducible suicide gene on a construct that would ablate the modified cells.

[0147] In accordance with the present disclosure, it was surprisingly found that NK cells provide a readily available cell to be engineered to contain at least one cytokine selecting from a group of cytokines including IL-15, IL-15 / IL-15sush, IL-15 / IL-15RA ( full length of IL-15 receptor a ), IL-15 / IL-15 anchor, IL-2, IL-7, IL-12, IL-18 and IL-21 polypeptide disclosed herein.

[0148] Allogeneic or autologous NK cells induce a rapid immune response but disappear relatively rapidly from the circulation due to their limited lifespan. Thus, applicants surprisingly discovered that there is reduced concern of persisting side effects using NK cell-based therapy.

[0149] According to one aspect of the present invention, NK cells can be transfected with cytokine polynucleotides and expanded in accordance to the present invention. NK cells can be derived from cord blood, peripheral blood, iPS cells and embryonic stem cells. According to one aspect of the present invention, NK-92 cells may be expanded and transfected with cytokine polynucleotides. NK-92 is a continuously growing cell line that has features and characteristics of natural killer (NK) cells (Arai, Meagher et al. 2008). NK-92 cell line is IL-2 dependent and has been proven to be safe (Arai, Meagher et al. 2008) and feasible. A pure population of NK-92 carrying the cytokine polynucleotide of interest may be obtained by sorting.

[0150] In some embodiments, the engineered cell includes an inducible suicide gene (“safety switch”) or a combination of safety switches, which may be assembled on a vector, such as, without limiting, a retroviral vector, lentiviral vector, adenoviral vector or plasmid. Introduction of a “safety switch” greatly increases safety profile. The “safety switch” may be an inducible suicide gene, such as, without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450. Other safety switches for elimination of unwanted modified NK or T-cells involve expression of CD20 or CD20 epitopes or CD52 or CD 19 or truncated epidermal growth factor receptor in T cells. All possible safety switches have been contemplated and are embodied in the present invention.

[0151] In one embodiment, the engineered cell includes a rituximab safety switch for elimination of unwanted modified immune cells. In a further embodiment, two rituximab binding sequences are incorporated to the hinge region of CAR.

[0152] In one embodiment, the engineered cell co-expresses a rituximab epitope expression construct with IL-15 / IL-15sushi through a peptide cleavage sequence selected from one of group of P2A, T2A, E2A and F2A. In a further embodiment, the rituximab epitope expression construct comprises of a signal peptide, two epitope domains of rituximab, CD8a hinge region and CD8a transmembrane domain.

[0153] Rituximab, originating as a CD20 targeted chimeric antibody, was developed by IDEC pharmaceuticals for treatment of malignancy.

[0154] Vector used for expressing a CAR

[0155] The polynucleotide described above can be cloned into a vector. A “vector” is a composition of matter which includes an isolated polynucleotide, and which can be used to deliver the isolated polynucleotide to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, phagemids, cosmids, and viruses. Viruses include phages, phage derivatives. Thus, the term “’vector'’ includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. In one embodiment, vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.

[0156] In an embodiment, the vector is a viral vector. In an embodiment, the viral vector is a retroviral vector or a lentiviral vector. In an embodiment, the engineered cell is virally transduced to express the polynucleotide sequence.

[0157] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the patient either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0158] Viral vector technology is well known in the art and is described, for example, in Sambrook et al, (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New' York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient and unique restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193). Lentiviral vectors have been well known for their capability of transferring genes into human T cells with high efficiency, but expression of the vector-encoded genes is dependent on the internal promoter that drives their expression. A strong promoter is particularly important for the third or fourth generation of CARs that bear additional co-stimulatory domains or genes encoding proliferative cytokines as increased CAR body size does not guarantee equal levels of expression. There are a wide range of promoters with different strength and cell-type specificity. Gene therapies using CAR T cells rely on the ability of T cells to express adequate CAR body and maintain expression over a long period of time. The EF- 1 a promoter has been commonly selected for the CAR expression.

[0159] ‘'Expression vector” refers to a vector including a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. The expression vector may be a bicistronic or multicistronic expression vector. Bicistronic or multicistronic expression vectors may include ( 1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; fusion of genes whose expressions are driven by a single promoter; (3) insertion of proteolytic cleavage sites between genes (self-cleavage peptide); and (iv) insertion of internal ribosomal entry sites (IRESs) between genes.

[0160] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor - 1 a (EF- 1 a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters, inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence, which is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metalothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0161] Expression of chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to. at least one of a SFFV (spleen-focus forming virus) or human elongation factor 11α (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor la (EF) promoter. Examples of less-strong / lower-expressing promoters utilized may include, but is not limited to, the simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or a part thereof. Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech Laboratories, Mountain View, CA) or a part or a combination thereof.

[0162] In a preferred embodiment, promoter is a MoMuL retroviral promoter in a retroviral vector. It has been unexpectedly discovered that MoMuL V promoter provides stronger expression and greater persistence in the transduced cells for CAR in accordance with the present disclosure.

[0163] The disclosure provides chimeric antigen receptor (CAR) compositions, methods of making and using thereof.

[0164] Generation of higher virus titer producing cell line for longer chimeric antigen receptor (CAR) vector constructs with multiple CAR and armored one or multiple cytokines The expression level of a retroviral or lentiviral vector can be significantly impacted by its size with larger vectors, particularly involving the expression of multiple units of CAR and armored one or multiple cytokines. Larger vectors generally result in a lower expression level due to limitations in the package capacity of retrovirus particle. In general, the more genetic material you try to package into a retrovirus, the less efficiently it can be produced and delivered to target cells, leading to reduced expression of the transgene of interest. These limitations present a significant bottleneck in the advancement of CAR-T cell therapies, particularly for constructs incorporating multiple CAR modules and cytokine armoring elements. The development of a high-titer retroviral packaging system optimized for the efficient delivery of large viral genome is crucial to overcoming these challenges and ensuring the robust, scalable production of clinically viable CAR-modified T cells. Additionally lower expression of CAR may affect CAR functions.

[0165] In some embodiments, inventors disclose the development of a retroviral packaging cell line for the efficient production of high-titer viral particles carrying longer chimeric antigen receptor (CAR) constructs. More particularly, the invention provides methods for generating stable, high- efficiency packaging cell lines, specifically utilizing the RD114 pseudotyped retroviral system, which is optimized to overcome limitations of conventional packaging systems that suffer from low viral titers when producing longer CAR constructs. The cell lines developed are capable of packaging and delivering the longer viral genome including multi-cistronic CAR constructs, that incorporate multiple CAR domains and armored cytokine components such as IL-15sushi and IL- 18. The invention further describes methodologies for large-scale clinical-grade production of high-titer stable viral particles that ensure efficient gene transfer into human immune cells including T and NIC cells with larger CAR constructs.

[0166] To address the challenges associated with reduced viral titers for large genetic constructs, the invention employs an enhanced retroviral transduction strategy'. A novel three -stage packaging approach is implemented as described below:

[0167] 1 ) high-titer virus particles are first generated using H29 cells;2) subsequently used to transduce RD114 packaging cells, thereby establishing stable viral producer cell lines; 3) cloning a high titer viral producing cell and then expansion of this viral producing single cell. Stage 1: Transfection of H29 cells and viral medium collection

[0168] To generate stable H29-based retroviral packaging cell lines, the H29 cells were first transfected using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) with a plasmid containing the CAR. Following transfection, viral supernatants were harvested at 40 hours and 64 hours post- transfection. filtered through a 0.22 pm filter, and used to transduce RD114 cells, leading to the generation of stable secondary retroviral packaging cell lines.

[0169] Stage 2: Transduction of the RD114 Cells

[0170] For RD114 transduction, viral supernatants harvested from H29 cells were used to infect RD114 cells in two rounds of transduction to maximize efficiency. The first-round transduction was performed at 50% confluence, followed by a second-round transduction 24 hours later. Transduced cells were selected via flow cytometry using VHH or F(Ab')2-PE labeling to confirm CAR expression, and high-expression single-cell clones were isolated using limiting dilution or FACS single cell sorting techniques.

[0171] Stage 3: Single Cell Screening:.

[0172] Two methods were utilized for isolating high virus-producing single-cell clones:

[0173] (i)Cell Monoclonal Screening Using Limited-Dilution Method

[0174] To generate stable, high-expression clones, a limited -dilution method was employed. Sorted RD114 CAR-expressing cells were diluted to an average of 0.5 cells per well in 48-well plates. Culture wells were observed daily under a 20xobjective microscope to identify wells containing a single cell-derived clone. These clones were labeled and cultured at 37°C and 5% CO2 for two weeks. A total of 22 single-cell clones were selected and expanded into larger wells. Flow cytometry analysis using goat anti-mouse F(Ab')2 or VHH antibody and streptavidin-PE was performed to confirm high CAR-T expression. The highest virus-producing clone was further expanded in T75 culture flasks, and viral supernatants were collected, filtered, and used to transduce activated human T cells. The highest transduction efficiency clone (S2) was selected, amplified, and frozen in multiple vials for master and working cell bank storage.

[0175] (ii)Cell Monoclonal Screening Using FACS-Aria Cell Sorter In addition to the limited-dilution method, single-cell isolation was performed using a FACS-Aria cell sorter. RD114 cells with the highest F(Ab')2 or VHH-PE expression were collected and sorted into individual wells of a 96-well plate, ensuring single-cell deposition. Any wells containing more than two cells were excluded on the first day of sorting. At least 20 single-cell clones were selected and monitored daily for two weeks using an Olympus CKX41 microscope (10xmagnification). The sorted clones were expanded in individual wells, and those reaching 70% confluence in a 96- well plate were transferred to 24-well plates. After two days, viral supernatants were collected and used to transduce activated human T cells. Flow cytometry analysis was conducted to identify the single RD114 clones with the highest T cell transduction efficiency. The selected high-expression clones were expanded, cryopreserved, and stored in liquid nitrogen for further use.

[0176] Using Stably Transduced RD114 Cell Supernatants for T Cell Transduction

[0177] Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed from liquid nitrogen storage, washed with T cell medium, and resuspended in AIM-V medium supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin. T cells were activated with IL-2 (300 lU / mL) and anti-CD3 antibody (50 ng / mL) and incubated at 37°C and 5% CO2 for 48 hours. Prior to transduction, untreated 12-well plates were coated with Retronectin (Takara, 7.5 pg / mL in DPBS) and incubated for 2 hours at room temperature. The coating solution was removed, and plates were blocked with DPBS containing 2% bovine serum albumin (BSA) for 30 minutes before initiating transduction.

[0178] Activated T cells were harvested, centrifuged, and resuspended at 1 x 10Λ6 cells / mL in T cell medium containing IL -2. Viral supernatants from RD114 cells were mixed with the cell suspension and added to Retronectin-coated plates. The plates were incubated at 37°C and 5% CO2 for 48 hours. Following incubation, transduced T cells were collected, centrifuged, and resuspended in fresh medium containing IL-2 for an additional 48 hours. The percentage of CAR expression on T cells was determined using flow cytometry analysis with anti-F(Ab')2-PE and CD3-PerCP, or anti- VHH-PE and CD3-PerCP antibodies. The data presented as examples show very high CAR expression efficacy using the methods described (Figure 18B, Figure, Figure 18B, Figure 20B, Figure 21B, Figure 22B , Figure 25B, Figure 27B and Figure 33C).

[0179] In an example showing in Figure 33C, the N399-20h-19N2N10-VAC ncCAR (N399) construct consists of two complete units of a humanized anti-CD20 single-chain variable fragment (scFv) CAR and a bi-epitope tandem CD19 nCAR (VHH1-VHH2) binds two distinct epitopes on CD19 antigen. Additionally, an IL-15 / IL-15sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence. As such a large sized armored two CARs, the CAR expression level was able to reach 68.89% in the T cells.

[0180] Experimental data disclosed in this invention, methods described were used to generate retroviral viruses expressing CARs.

[0181] Methods for generating CAR T or CAR NK cells

[0182] 1) Methods for Isolation T and NK cells:

[0183] Peripheral blood mononuclear cells (PBMCs) were isolated from patient-derived buffy coat samples using Ficoll-Paque density gradient centrifugation. The buffy coat was diluted with an equal volume of phosphate-buffered saline (PBS) and carefully layered over Ficoll-Paque solution in a 50 mL conical tube. The samples were centrifuged at 400 x g for 30 minutes at room temperature without a break. After centrifugation, the PBMC layer was collected and washed twice with PBS to remove residual platelets and Ficoll. The isolated PBMCs were resuspended in AIM V medium supplemented with 10% fetal bovine serum (FBS), 300 lU / mL IL-2, 2 mM L- glutamine, and 100 U / mL penicillin-streptomycin for further processing. The buffy coats were then counted using a hemocytomctcr and cultured at a density of 0.5 x 106cclls / mL prior to CAR transduction.

[0184] Fresh human umbilical cord blood was obtained and processed within 24 hours of collection. The cord blood was diluted 1 : 1 with PBS and carefully layered over Ficoll-Paque solution. The samples were centrifuged at 400xg for 30 minutes at room temperature without a brake to separate mononuclear cells. The mononuclear cell layer was collected and washed twice with PBS to remove any residual Ficoll. To isolate NK ceils, the mononuclear cells were subjected to negative selection using the EasySep Human NK Cell Isolation Kit (STEMCELL Technologies) according to the manufacturer’s instructions. The purified NK cells were resuspended in human NK MACS medium (Miltenyi Biotec) supplemented with 5% human AB serum, 2 mM L -glutamine, 500 lU / mL IL-2, and 10 ng / mL recombinant human IL-15. The cells were then cultured at 37°C in 5% CO2 at a density of 0.7 x 106cells / mL to promote NK cell viabi 1 i ty and expansion.

[0185] 2) T Cell Transduction with CAR Construct retroviruses

[0186] For CAR transduction, purified T cells were first activated with 50 ng / mL anti-human CD3 antibody (Miltenyi) and cultured at 37°C and 5% CO2 for 48 hours.

[0187] Retroviral supernatants encoding the respective CAR constructs were generated using high virus titer producing RD 114 cell line. Viral particles were harvested after 48 hours incubation. The viral supernatants were concentrated by 1,500 x g for 5 minutes at 4°C.

[0188] For transduction, pre-activated T cells were plated at a density of 5 x 105cells per well in non- tissue culture-treated 24-well plates coated with RetroNectin (Takara, 7.5 pg / mL in DPBS). Activated T cells were then mixed with 1 ml of viral supernatant from RD114 cells. After viral transduction, T cells were incubated at 37°C for 48 hours before removing the viral supernatant. Following transduction, CAR-T cells were expanded in AIM V medium supplemented with IL-2 (300 TU / mL) to promote T cell proliferation and survival. The cells were maintained at a density of 1 x 106cclls / mL, with fresh media replenishment every 2-3 days.

[0189] Cell expansion was measured at various time points using trypan blue exclusion staining and hemocytometry to determine viable cell counts.

[0190] The efficiency of CAR transduction was evaluated five days post-transduction using flow cytometry by staining the cells with anti-VHH and anti-CD3, or anti-F(ab’)2 and anti-CD3 antibodies to confirm CAR expression. 3) NK Cell Transduction with CAR Constructs

[0191] For NK cell transduction, Retroviral supernatants encoding the respective CAR constructs (GI, GIV, GI18, and GIVI 8) were generated using high virus titer producing RD114 cell line. Viral particles were harvested after 48 hours incubation. The viral supernatants were concentrated by 1,500 x g for 5 minutes at 4°C.

[0192] For transduction, isolated NK cells were plated at a density of 5 x 105cells per well in non-tissue culture-treated 24-well plates coated with RetroNectin (Takara, 7.5 pg / mL in DPBS). NK cells were then mixed with 1 ml of viral supernatant from RD114 cells. After viral transduction, NK cells were incubated at 37°C for 48 hours before removing the viral supernatant.

[0193] Following transduction, NK cells were expanded in human NK MACS medium (Miltenyi Biotec) supplemented with 5% human AB serum, 2 mM L-glutamine, 500 lU / mL IL-2, and 10 ng / mL recombinant human IL-15.

[0194] Cell expansion was measured at various time points using trypan blue exclusion staining and hemocytometry to determine viable cell counts.

[0195] CAR expression was assessed five days post-transduction using flow cytometry' with anti-VHH and anti-CD56, or anti-F(ab’)2 and anti-CD56 antibodies, confirming efficient CAR expression in transduced NK cells.

[0196] Co-culture killing assay using CAR T or NK can be performed using a standard method as described (Leukemia 2018 Jun;32(6): 1317-1326)

[0197] BCMA nanobodv CAR (“BCMA nCAR”)

[0198] B-Cell Maturation Antigen (BCMA), otherwise known as TNFRSF17 or CD269 is a non- glycosylated type III transmembrane protein belonging to the superfamily tumor necrosis receptors. BCMA is expressed on the surface of differentiated plasma cells and is thereby a cell surface receptor. It is involved in the survival and development of B-cells. In further embodiments, BCMA nCAR in a T or NK cell may be used to eradicate or kill BCMA+ population. In a further embodiment, the disclosed disclosure comprises methods and compositions of deleting BCMA+ populations in multiple myeloma.

[0199] In further embodiments, BCMA nCAR in a T or NK cell may be used to eradicate or kill BCMA+ population. In a further embodiment, the disclosed disclosure comprises methods and compositions of deleting the BCMA + population associated with autoimmune diseases.

[0200] BCMA-CD19 nanobody antibody compound CAR (“BCMA-CD 19 ncCAR”)

[0201] While killing multiple myeloma cells can provide short-term relief, LSCs (myeloma leukemic stem cells), if not destroyed, will always re-grow, causing the patient to relapse. It is imperative that LSCs be destroyed to achieve durable cures for multiple myeloma disease. Without wishing to be bound by theory, it is believed that a small subset of multiple myeloma cells is stem cells that are CD19 positive and associated with disease progression and relapses, and a bulky myeloma cell population is BCMA positive. Therefore, it is critical to develop new therapies that can specifically target both the myeloma stem cell population and the bulky myeloma population. A compound CAR in the present disclosure targets BCMA+ and / or CD 19+ positive populations of multiple myeloma cells and is embodied herein.

[0202] In some embodiments, the present disclosure provides a method of eradicating or killing myeloma stem cells (LSCs) or bulk myeloma cells expressing CD 19 and / or BCMA. In this embodiment, a T or NK engineered cell having a BCMA unit and a CD 19 unit is administered to a patient in need thereof.

[0203] In some embodiments, the disclosed disclosure comprises methods and compositions of deleting both BCMA and CD 19 populations in multiple myeloma to prevent relapses using a BCMA-CD 19 ncCAR. CAR is more powerful in eliminating myeloma cells when combination of two units of BCMA and CD 19 (BCMA-CD 19) together in a vector or a cell.

[0204] In further embodiments, a compound CAR, BCMA-CD 19 ncCAR in a T or NK cell may be used to eradicate or kill BCMA+CD19+ or BCMA+CD19- or BCMA-CD 19+ populations. In some embodiments, CD 19+ populations can be early precursors for multiple myeloma cells, and CD19-BCMA+ cells can be more differentiated malignant multiple myeloma cells. In some embodiments, the disclosed invention comprises methods and compositions of deleting both early precursor of multiple myeloma cells and more differential malignant multiple myeloma cells using a BCMA-CD19 ncCAR (a version of BCMA-CD19 cCAR using camelid derived antibodies) T or NK cell. In a further embodiment, the disclosure comprises methods and compositions of targeting both early precursor and more differential malignant cells to completely eliminate malignant clones for multiple myeloma using a BCMA-CD19 cCAR T or NK cell.

[0205] The present disclosure further discloses a compound CAR construct with enhanced potency of anti-myeloma cell activity against cells co-expressing target antigens, and yet retains sensitivity to tumor cells only expressing one antigen. In addition, each CAR of the compound CAR includes one or two co-stimulatory domains and exhibits potent killing capability in the presence of the specific target.

[0206] Without wishing to be bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with BCMA-CD19 ncCAR (Figure 16, Figure 17 and Figure 22) provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target myeloma cells or recruiting innate immune cells to myeloma cells.

[0207] Without wishing to be bound by theory , it is believed that co-expression of IL-15 / IL- 15 sushi or IL- 18 or combination with BCMA-CD 19 ncCAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target myeloma cells or recruiting innate immune cells to myeloma cells.

[0208] In some embodiments, a BCMA-CD 19 ncCAR targets cells expressing BCMA or CD 19 antigens or both. The targeted cells may be cancer cells, such as, without limiting, lymphomas, or leukemias or plasma cell neoplasms. In further embodiments, plasma cell neoplasms are selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom’s macroglobulinema, heavy chain diseases, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.

[0209] Without wishing to be bound by theory, it is believed that co-expression of IL-21 or IL-IL-21 anchor with BCMA-CD19 ncCAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target myeloma cells or recruiting innate immune cells to myeloma cells.

[0210] In some embodiments, the disclosure provides a method of depleting B cells, immature B cells, memory B cells, plasmablasts, long lived plasma cells, or plasma cells in patients with an autoimmune disease by administering to patients CAR or compound CAR (BCMA-CD19 ncCAR) T cells or NK cells.

[0211] BCMA-CD19 ncCAR targeted cells are B cells, immature B cells, memory B cells, plasmablasts, long lived plasma cells, or plasma cells in patients with autoimmune diseases. The autoimmune diseases include systemic scleroderma, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases (such as Crohn’s disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjorgen's syndrome, myasthenia gravis, neuromyelitis optica, polymyositis, pulmonary alveolar proteinosis, granulomatosis and vasculitis, Addison's disease, antigen-antibody complex mediated diseases, antiphospholipid syndrome and anti-glomerular basement membrane disease.

[0212] BCMA-CDI9 ncCAR targeted cells are B cells, immature B cells, memory B cells, plasmablasts, long lived plasma cells, or plasma cells in patients with autoimmune diseases. Examples of autoimmune diseases include but are not limited to, achalasia, Addison’s disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4- activating rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), diabetes mellitus, discoid lupus. Dressier’ s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, Hashimoto’s thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), hypogammalglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4- related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, membranous nephropathy, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage- Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s syndrome, reactive Arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, sensitized / preformed antibodies in solid organ transplant, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener’s disease. Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorders (MOGSD), neuromyelitis optica spectrum (NMOSD), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In preferred embodiments, the autoimmune disorder is not IgG4-reiated disease. In preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV), SLE (e.g. relapsed or refractory SLE), or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV) or rheumatoid arthritis (e.g. relapsed or refractory' rheumatoid arthritis).

[0213] In some embodiments, immune cells including B cells, immature B cells, memory / B cells, plasmablasts, long lived plasma cells, or plasma cells in patients with autoimmune diseases can be eliminated by a BCMA or CD 19 bispecific nanobody antibody as a method previously described (Vaccines (Basel). 2022 Aug 17;10(8):1335). Bispecific nanobody antibodies can be constructed from two nanobodies targeting different targets of cells by genetic engineering to improve the specificity of antitumor or anti-immune cell antibodies.

[0214] In some embodiments, BCMA and CD 19 CAR nucleic acid sequences can be incorporated in the same vector expression and expression is controlled by their own promoters. In some embodiments, BCMA CAR T or NK cells, and CD 19 CAR T or NK cells can be generated separately and then administrate to a host sequentially.

[0215] In another embodiment, the present disclosure provides a method of treating an autoimmune disease. An autoimmune disorder is selected from a group of diseases including autoimmune disease comprises systemic lupus erythematosus (SLE), multiple sclerosis (MS), Inflammatory bowel disease (IBD), Rheumatoid arthritis, Sjogren syndrome, dermatomyosities, autoimmune hemolytic anemia, Neuromyelitis optica (NMO), NMO Spectrum Disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutorphil cytoplasmic autoantibodies (ANCAs) associated with systemic autoimmune small vessel vasculitis syndromes or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener’s granulomatosis, Pemphigus vulgaris (PV) and pemphigus foliaceus (PF).

[0216] An organ transplant represents a new life for a person and organs that can be transplanted could include the kidneys, heart, lungs, pancreas and intestine. However, many patients are unable to receive a potentially life-saving organ because of pre-existing or developing donor-specific antibody against the donor’s antigens such human leukocyte antigens (HLA). Thus, patients may lose the donated organ. Currently there are few treatment options available for antibody mediated rejection, and an enormous unmet need in the field for efficacious treatment of antibody mediated rejection. Deletion of B cells or plasma cells or both using CAR T / 'NK cell provide a therapy for antibody-mediated rejection.

[0217] BCMA-CD19 ncCAR targeted cells are B cells, immature B cells, memory B cells, plasmablasts, long lived plasma cells, or plasma cells in patients with the antibody-mediated rejection associated with organ rejections.

[0218] CD 19 nCAR (CD 19 nanobody CAR)

[0219] CD 19 is a cell-surface antigen found in most B- malignancies including B cell acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia / lymphoma (CLL / SLL)), and Non-Hodgkin lymphomas, making is an attractive target for engineering targeted CAR-T cells. CARs rely on classical single-chain fragment variable (scFv) with light and heavy chains connected by a peptide linker. This linker may induce immunogenicity in humans. FDA approved CD 19 CAR uses the murine derived scFv FMC63 clone which has demonstrated induction of neutralizing antibodies against the CAR after infusion, impairing the potential to re-dose the patient. Additionally, the scFv structure may exhibit instability when binding to the antigen, and result in T cell exhaustion. To address such limitations, a novel CD 19 nanobody antibody CAR (CD 19 nCAR) construct utilizing camelid-derived nanobodies that target B cells and lymphoma or leukemia expressing CD 19 is produced.

[0220] In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a CD 19 nCAR (nanobody antibody CAR) polypeptide against a cell expressing CD 19.

[0221] In one embodiment, the present disclosure provides a method of treating leukemia or lymphoma expressing CD 19 in a patient by administering a CD 19 nCAR engineered cell to a patient in need thereof.

[0222] In further embodiments, the CD 19 nCAR engineered cells target cells having cell surface CD 19 antigens. In another embodiment, the targeted cells are malignant B cell lymphoma / leukemia such as, without limiting, B-ALL (B cell acute lymphoblastic leukemia) / B-LBL (B cell lymphoblastic lymphoma), high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.

[0223] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a CD 19 nCAR (nanobody antibody CAR) polypeptide against cells expressing CD 19. In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a CD 19 nCAR (nanobody antibody CAR) polypeptide to deplete autoantibody producing cells, such as B cells, immature B cells, memory B cells and plasmablasts associated with autoimmune diseases

[0224] Without wishing to be bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with CD 19 nCAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target CD19 positive leukemia / lymphoma cells or recruiting innate immune cells to tumor cells.

[0225] Without wishing to be bound by theory', it is believed that co-expression of IL-15 / IL-15sushi or IL- 18 or combination with CD 19 nCAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target leukemia / lymphoma cells or recruiting innate immune cells to tumor cells.

[0226] In some embodiments, the disclosure provides a method of depleting B cells, immature B cells, memory B cells and plasmablasts in patients with an autoimmune disease by administering to patients CAR T cells or NK cells.

[0227] In an embodiment, the present disclosure provides a method of treating an autoimmune disease, said method including administering a CD 19 nCAR engineered cell described above to a patient in need thereof; wherein said autoimmune disease comprises systemic lupus erythematosus (SLE), multiple sclerosis (MS), Inflammatory bowel disease (IBD), Rheumatoid arthritis, Sjogren syndrome, dermatomyosities, autoimmune hemolytic anemia, Neuromyelitis optica (NMO), NMO Spectrum Disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutorphil cytoplasmic autoantibodies (ANCAs) associated with systemic autoimmune small vessel vasculitis syndromes or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), or eosinophilic granulomatosis with polyangiitis (EGPA, Churg- Strauss syndrome). In another embodiment, the present disclosure provides a method of treating asthma. The method includes administering an engineered cell described above.

[0228] In another embodiment, the present disclosure provides a method of treating organ rejection. The method includes administering an engineered cell described above.

[0229] In another embodiment, the present disclosure provides methods of CD 19 nCAR T cells for treating B-cell malignancies including but not limited to B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (TALL), Acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK-positive large B-cell lymphoma, Burkitt's lymphoma. Chronic Myelogenous Leukemia (CML), Chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma Classical Hodgkin lymphoma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Intravascular large B-cell lymphoma, Large B-cell lymphoma arising in HHV8-associated multicentric Castleman's disease, Lymphomatoid granulomatosis, multiple myeloma, myelodysplasia and myelodysplastic syndrome, Lymphoplasmacytic lymphoma, Mantle cell lymphoma (MCL), Marginal zone B-cell lymphoma (MZL), Mucosa-Associated, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, Lymphatic Tissue lymphoma (MALT), Nodal marginal zone B cell lymphoma (NMZL), Nodular lymphocyte predominant Hodgkin's lymphoma, Non-Hodgkin's lymphoma, Plasmablastic lymphoma, Plasmacytoid dendritic cell neoplasm Primary central nervous system lymphoma. Primary effusion lymphoma, Splenic marginal zone lymphoma (SMZL), and Waldenstrom’s macroglobulinemia. In some embodiments, the B-cell lymphoma is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), Chronic lymphocytic leukemia (CLL), Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma (MCL), Marginal zone B-cell lymphoma (MZL), Mucosa-Associated Lymphatic Tissue lymphoma (MALT), and Non-Hodgkin’s lymphoma. In some embodiments, the B -cell lymphoma is Non-Hodgkin’ s lymphoma and "preleukemia" which are a collection of hematological conditions united by dysplasia of myeloid blood cells, and to disease associated with CD19 expression include, but not limited to atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing CD 19, autoimmune conditions and combinations thereof.

[0230] In another embodiment, the present disclosure provides methods of CD 19 nCAR T cells for treating autoimmune diseases including but not limited to achalasia, Addison’s disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4- activating rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), diabetes mellitus, discoid lupus, Dressier' s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, Hashimoto’s thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein puipura (HSP), herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), hypogammalglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4- related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease. Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus. Lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, membranous nephropathy, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage- Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Rajmaud’s syndrome, reactive Arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RES), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, sensitized / preformed antibodies in solid organ transplant, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), systemic lupus erythematosus (SEE), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener’s disease. Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorders (MOGSD), neuromyelitis optica spectrum (NMOSD), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren’s syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In preferred embodiments, the autoimmune disorder is not IgG4-related disease. In preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV), SLE (e.g. relapsed or refractory SLE), or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV) or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis).

[0231] Bi-epitope tandem CD 19 nCAR

[0232] Bi-epitope tandem CAR has a dual domain to bind two different parts of the antigen within one Bi-CAR. The structure of a dual domain is formed with scFvl-scFv2 or VHH1-VHH2. Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain( s) and CD3 zeta chain.

[0233] In one embodiment, Bi-epitope tandem CAR can be two particular antigen recognition sites are joined by a peptide linker, placed in tandem on a single intracellular domain and expressed as a single CAR on a cell surface.

[0234] In a particular embodiment, antigen recognition domain includes two different heavy chain domains (VHH). Each heavy chain domain binds to a different epitope of the same antigen, which can enhance the function or prevent from antigen escape associated with disease relapse.

[0235] In one embodiment, Bi-epitope tandem CD 19 CAR bears a leader, two CD19 antigen recognition domains including two different VHHs (each VHH domain binds to a different epitope of the same antigen, CD19), a hinge region, a transmembrane domain, at least one co-stimulatory domain, CD3 signaling domain.

[0236] In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a bi-epitope tandem CAR polypeptide against a cell expressing CD 19.

[0237] In one embodiment, the disclosure provides a method of Bi-epitope tandem CAR to treat diseases as same as those of CD 19 nCAR described above.

[0238] In one embodiment, the disclosure provides a method of Bi-epitope tandem CAR to treat diseases as same as those of CD 19 nCAR described above. Bi-epitope tandem CD19 CAR T cells targeted cells are B cells, immature B cells, memory B cells and plasmablasts associated with autoimmune diseases described above or antibody-mediated rejection associated with organ rejections

[0239] 11CD20-CD19 ncCAR

[0240] Initial remission of most B-ALL can be seen in CD 19 CAR T therapy but relapses with epitope loss occur in 10% to 20% of responders. Therefore, a single target for CAR based treatment may not be sufficient to prevent leukemia relapse.

[0241] In one embodiment, the antibody recognition domain includes the binding variable region of a humanized monoclonal antibody, single chain fragment variable (scFv) for CD20 (11CD20). The scFv includes one light and heavy of antibody. In a particular embodiment, antigen recognition domain is composed of the second recognition domain, camelid VHH for CD 19. Each recognition domain binds to a different epitope of different antigens.

[0242] In some embodiments, the compound CAR (ncCAR) targets different antigens.

[0243] In a particular embodiment, antigen recognition domain is composed of two different heavy chain domains (VHH), VHH1-VHH2 for CD 19 called CD 19 bi-epitope tandem CAR. Each VVH domain binds to a different epitope of the same antigen for the avoidance of relapses due to the epitope loss. A VHH antibody is more stable and robust than a whole antibody. Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain.

[0244] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a 11CD20-CD19 ncCAR or Bi-epitope tandem CD19CAR polypeptide to deplete autoantibody producing cells, such as B cells, immature B cells, memory B cells and plasmablasts associated with autoimmune diseases as described above. In another embodiment, the present disclosure provides methods of hCD20-CD19 ncCAR or Bi- epitope tandem CD 19 CAR T cells for treating autoimmune diseases including but not limited to achalasia, Addison’s disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti- GBMy'anti-TBM nephritis, anti-PAD4-activating rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (A1ED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory' demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), diabetes mellitus, discoid lupus, Dressier’ s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, Hashimoto’s thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), hypogammalglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, membranous nephropathy, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis. myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s syndrome, reactive Arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RES), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, sensitized / preformed antibodies in solid organ transplant, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), systemic lupus erythematosus (SEE), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener’s disease. Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorders (MOGSD), neuromyelitis optica spectrum (NMOSD), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In preferred embodiments, the autoimmune disorder is not IgG4-related disease. In preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV), SLE (e.g. relapsed or refractory SLE), or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV) or rheumatoid arthritis (e.g. relapsed or refractory' rheumatoid arthritis). In one embodiment, the present disclosure provides a method of treating leukemia or lymphoma expressing CD 19 or CD20 or both in a patient by administering a CD 19 nCAR engineered cell to a patient in need thereof.

[0245] In further embodiments, the 11CD20-CD19 ncCAR or Bi-epitope tandem CD 19 nCAR engineered cells target cells having cell surface CDI9 or CD20 antigens. In another embodiment, the targeted cells are malignant B cell lymphoma / leukemia such as, without limiting, B-ALL (B cell acute lymphoblastic leukemia) / B-LBL (B cell lymphoblastic lymphoma), high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.

[0246] Without wishing to be bound by theory, it is believed that co-expression of IL-15 / IL-15sushi or IL- 18 or combination with 11CD20-CD19 ncCAR or Bi-epitope tandem CD 19 nCAR provides long-term durable remission in patients by increasing the sensitivity' of CAR recognition of target cells or recruiting innate immune cells to leukemia / lymphoma cells.

[0247] Claudin 18.2 nCAR

[0248] The Claudin 18 protein has two isoforms, namelyy Claudin 18.1 and Claudin 18.2

[0249] Claudin 18.2 amino acid sequence: Claudin 18.1 is predominately present in the lung epithelia while Claudin 18.2 is predominately present in the stomach epithelium. Additionally, Claudin 18.2 is highly expressed in primary malignant tumors such as gastric, breast, colon and liver cancers. Claudin 18.2 has thus become an exciting target to treat solid tumors including gastric and pancreatic cancers.

[0250] CLDN18.2 specific antibodies are difficult to produce due to its high homology to CLDN18.1. There are only 7-8 amino acid differences between these two splice variants in the first extracellular loop that makes the generation of this antibody difficult. The combination of DNA transfection, protein as immunogens coupled with high-throughput FACS-based screening approaches is adopted to isolate and identify CLDN 18.2 VHH. In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a Claudin nCAR (nanobody antibody CAR) polypeptide against a cell expressing Claudin 18.2.

[0251] In further embodiments, the Claudin 18.2 nCAR engineered cells target cells having cell surface Claudin 18.2 antigens. In another embodiment, the targeted cells are cancers including stomach carcinoma, esophagus carcinoma, bladder carcinoma, breast carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal carcinoma, gastric sarcoma, glioma, lung carcinoma, melanoma, , osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, a head, a neck tumor, and a solid tumor.

[0252] It was surprisingly discovered that co-expression of IL-15 / IL-15sushi or IL-18 or combination with Claudin 18.2 nCAR provides CAR cell expansion and killing of targeted cells.

[0253] It was also surprisingly discovered that co-expression of IL-15 / IL-15sushi and IL- 18 provides a synergistic efficacy in killing targeted cells.

[0254] In another embodiment, the present disclosure provides CLDN-18.2 targeted therapy. The therapy could include the administration of a nuclei acid such as an RNA or mRNA encoding a Claudin 18.2 antibody or a Claudin 18.2 CAR. The therapy could also include synergistic therapeutic with chemotherapeutics agents or a Claudin 18.2 enhancing agent.

[0255] CLL1-CD33 ncCAR

[0256] In one aspect of the present disclosure, CLL-1 antigen is one of the targets for cCAR therapy. C- type lectin-like- 1 (CLL-1, also Called CLL1) is also known as MICE, CLEC12A, CLEC-1 and DCAL2.

[0257] CLL-1 expression is seen in acute myeloid leukemia (AML) blasts and leukemic stem cells.

[0258] CLL-1 is expressed in a variety of leukemias including myelomonocytic leukemia (M4), acute monocytic leukemia (M5), acute promyelocytic leukemia (M3), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms and myelodysplastic syndromes (MDS).

[0259] CLL-1 is expressed on a subset of leukemic cells related to leukemic stem cells (LSCs), the ablation of which is essential in preventing disease refractoriness and relapse.

[0260] In one aspect of the present disclosure, CD33 antigen is one of the targets for cCAR therapy. CD33 is a transmembrane receptor expressed on 90% of malignant cells in acute myeloid leukemia. Thus, according to the present disclosure, CLL-1 and CD33 target antigens are particularly attractive from a safety standpoint.

[0261] In an embodiment, the CLL1-CD33 ncCAR engineered cells target cells having cell surface CLL1 or CD33 or both antigens. In another embodiment, the targeted cells are leukemia including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, myelomonocytic leukemia, and myelodysplastic syndromes (MDS).

[0262] In an embodiment, co-expression of IL-15 / IL-15sushi or IL-18 or combination with CLL1-CD33 ncCAR provides CAR cell expansion and killing of targeted cells.

[0263] In a further embodiment, co-expression of IL-15 / IL-15sushi and IL-18 provides a synergistic efficacy in killing targeted cells.

[0264] In another embodiment, the present disclosure provides CLL1-CD33 ncCAR targeted therapy. The therapy could also include the combination therapy, such as chemotherapeutics agents and others described below.

[0265] Combination therapy

[0266] The combination therapy with CAR approach can provide more personalized treatments that are tolerable and don’t affect quality of life. Combination therapy can reduce the potential for drug resistance for cancers and autoimmune diseases. In one embodiment, CAR treatments for cancer and autoimmune diseases can be combined with other therapies including surgery, chemotherapy, radiation, immunotherapy, biologic agents, immunosuppressing agents (for autoimmune diseases) and monoclonal antibody therapies.

[0267] Immune cells or immunomodulatory cells including, but not limited to, T cells, macrophage, NK cells and NK T cells have been used for treatment of autoimmune disorders, autoreactive cells targeted to cells, tissues, and organs, infectious diseases, and cancers. In the preferred embodiment, T-cells engineered to express CAR unit or units targeted toward disease have indicated profound specificity and cytotoxicity. In another embodiment, immune cells or immunomodulatory cells including but not limited to T-cells, macrophage, NK cells and dendritic cells are engineered to express CAR unit or units targeted toward disease have indicated profound specificity and cytotoxicity.

[0268] In one embodiment, the engineered CAR-T cells are administered in combination with an agent that increases the efficacy of CAR T-cell therapy, including but not limited surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, stem cell or bone marrow transplant and / or hormone therapy.

[0269] T cell engagers are characterized by acting as a bridge between an antigen on a target cell and different antigen on a T cell (Ruehls A M et al. Immunol Cell Biol 2015; 93(3): 290-296).

[0270] T cell engagers are constructed of two antigen binding domains connected in tandem by a flexible linker. One antigen binding domain binds to a T cells, usually CD3, while the second antigen binding domain binds to a tumor associated antigen or an immune cells associated antigen. The antigen binding domain could be scFv or VHH. The construct allows to bring T cells close to the targeted tumor cells resulting stimulating T cell activation, targeted killing and cytokine production. The second antigen recognition domain are selected from the group consisting of Claudin 18.2, CD19, CD20, CD7 and BCMA. In one embodiment, the engineered CAR-T cells or nanobody antibody or T cell engagers are administered in combination with an agent that ameliorates one or more side effects associated with the administration of CAR T-cell therapy...

[0271] A chimeric antigen receptor (CAR) polypeptide includes a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

[0272] The "antigen recognition domain" includes a polypeptide that is selective for or targets an antigen, receptor, peptide ligand, or protein ligand of the target, or a polypeptide of the target.

[0273] The antigen recognition domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. The antigen recognition domain may include a portion of 1g heavy chain linked with a portion of Ig light chain, constituting a single chain fragment variable (scFv) that binds specifically to a target antigen. The antibody may be monoclonal or polyclonal antibody or may be of any type that binds specifically to the target antigen. In another embodiment, the antigen recognition domain can be a receptor or ligand. In particular embodiments, the target antigen is specific for a specific disease condition and the disease condition may be of any kind as long as it has a cell surface antigen, which may be recognized by at least one of the chimeric receptor constructs present in the compound CAR architecture. In a specific embodiment, the chimeric receptor may be for any cancer for which a specific monoclonal or polyclonal antibody exists or is capable of being generated. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia have antigens specific for the chimeric receptors.

[0274] In some embodiments, antigen recognition domain can be non-antibody protein scaffolds, such as but not limited to, centyrins, non-antibody protein scaffolds that can be engineered to bind a variety of specific targets with high affinity. Centyrins are scaffold proteins based on human consensus tenascin FN3 domain, are usually smaller than scFv molecules CAR molecules.

[0275] The target specific antigen recognition domain preferably includes an antigen binding domain derived from an antibody against an antigen of the target, or a peptide binding an antigen of the target, or a peptide or protein binding an antibody that binds an antigen of the target, or a peptide or protein ligand (including but not limited to a growth factor, a cytokine, or a hormone) binding a receptor on the target, or a domain derived from a receptor (including but not limited to a growth factor receptor, a cytokine receptor or a hormone receptor) binding a peptide or protein ligand on the target.

[0276] In one embodiment, the antigen recognition domain includes the binding moiety or variable region of a monoclonal or polyclonal antibody directed against (selective for) the target.

[0277] In another embodiment, the antigen recognition domain includes camelid single domain antibody, or portions thereof. In one embodiment, camelid single- domain antibodies include heavy-chain antibodies found in camelids, or VHH antibody. A VHH antibody of a camelid (for example camel, dromedary, llama, and alpaca) refers to a variable fragment of a camelid single-chain antibody (See Nguyen et al, 2001 ; Muyldermans, 2001), and also includes an isolated VHH antibody of camelid, a recombinant VHH antibody of camelid, or a synthetic VHH antibody of camelid.

[0278] In another embodiment, the antigen recognition domain includes the binding variable region of a monoclonal antibody, single chain fragment variable (scFv). The scFv includes one light and heavy of antibody. In a particular embodiment, antigen recogniztion domain includes tw'o different heavy chain domains (VHH). Each heavy chain domain binds to a different epitope of the same antigen or different antigen. In one embodiment, the antigen recognition domain includes a single heavy chain domain.

[0279] Immunomodulator(s)

[0280] The present disclosure includes methods of improving CAR T / NK cell expansion, persistency and anti-target activity by co-expressing secretory IL-15 / IL-15sushi complexes in an expression cassette (see Figure 16A and Figure 16B) . In a further embodiment, engineered CAR T / NK cells comprise secretory 1L-15 / IL-I5sushi (also called IL15 / IL15sushi) complex, which can promote expansion of specific CAR T / NK cells, and promote infiltrate of innate immune cells to the target sites resulting in greater destruction.

[0281] IL- 15 is a pleiotropic cytokine that is associated with a huge range of immunology and plays an important role in both adaptive and innate immunity. A 65 amino acid sequence of the extracellular portion of IL-15Ra, called sushi domain involves the binding of IL-15,

[0282] Prior to the art, it is known that IL-15 has a short biological half-life. The sushi domain is incorporated to increase IL- 15 half-life up to ten-fold by forming an IL-15 / IL-15sushi complex, leading to longer persistency.

[0283] In the prior art, constitutive expression of a high level of IL- 15 in mice could cause leukemia (Fehniger et al, J Exp Med. 2001 Jan 15 ; 193(2):219-31 ). Therefore, this leukemia matter with IL- 15 could teach away a skilled person to generate a more powerfol version of IL- 15 with a longer biological half-life and longer persistency to arm a CAR.

[0284] It was surprisingly found that only picogram quantities of IL-15 / IL-15sushi (Figure 31A) in mice and in patients ( Front Immunol 2022 Sep 12: 13:997482; Rev Rep 2021 Apr;17(2):652-661 ) were produced by infused CAR T cells co-expressing IL-15'IL-15sushi, and there is no evidence of autonomous growth or leukemic transformation in human clinical trials after an over 3 -year observation.

[0285] In accordance with the present disclosure, the inventors have also found that immune cells transduced with secreted IL-15 / IL-15sushi are superior in persistency and immunity-inducing effect to the conventional immune cells in vivo.

[0286] BCMA-CD19 ncCAR target cells expressing BCMA or CD 19 or both

[0287] Alpacas were immunized with human BCMA or CD 19 protein. Peripheral blood mononuclear cell (PBMC) mRNA was isolated and processed into a VHH gene library, transformed into phage- competent bacteria to generate a bacteriophage library. This library was panned against BCMA or CD19 antigens to enrich for binding clones, identified by high-throughput ELISA and cellular binding by flow cytometry. VHH candidates with binding greater than two-fold above background were sequenced. Using Biolayer Interferometry (BLI), association and dissociation rates were determined. Selected nanobodies were used to create a novel construct, termed BCMA-CD19 nanobody compound CAR (ncCAR). BCMA-CD19 ncCAR construct bears complete units of CARs. Each consists of the leader sequence, VHH nanobody antibody, hinge region, transmembrane domain, co-stimulatory domain and CD3 zeta chain signaling domain. Optionally, an enhancer, IL-15 / IL-15sushi is separated from the first CAR and second CAR by a second cleavage site that flanks either of the two distinct CAR units. Methods of characterization of BCMA-CD19 ncCARcan be seen in the references (Li et al, Nat Commun. 2023 Sep 22; 14(l):5920; Chen et al. Leukemia. 2018 Feb; 32(2): 402^112).

[0288] BCMA-CD19 ncCAR T demonstrated potent and selective cytotoxicity against BCMA or CD 19- positive cells in vitro. Co-culture assays revealed significant BCMA or CD19 positive cell lysis, indicating effective dual-antigen targeting. In mouse models, both nCAR and ncCAR showed substantial cytotoxic activity, with depletion of BCMA and CD19 positive cells and significant durable persistency. This ncCAR T represents a promising therapeutic strategy for patients with autoimmune diseases or malignancies who relaspse after CAR therapy. The novel construct may provide improved efficacy and potential to re -dose a CAR relapsed patient.

[0289] In one embodiment, the present disclosure provides an engineered nanobody antibody compound CAR (ncCAR) -T cell comprised a first chimeric antigen receptor polypeptide including a first antigen recognition domain (VHH), a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and a second chimeric antigen receptor polypeptide including a second antigen recognition domain (VHH), a second signal peptide, a second hinge region, a second transmembrane domain, a second co- stimulatory domain, and a second signaling domain; wherein the first antigen recognition domain and the second antigen recognition domain are different from each other as they are specific to different antigens; and second antigen recognition domain are selected from the group consisting of BCM A and CD 19

[0290] In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ncCAR polypeptide against a cell expressing CD19 or BCMA or both.

[0291] In one embodiment, the present disclosure provides a method of treating leukemia or lymphoma or myeloma expressing CD19 or BCMA or both in a patient by administering a BCMA-CD19 ncCAR engineered cell to a patient in need thereof. In further embodiments, the BCMA-CD19 ncCAR engineered cells target cells having cell surface CD 19 or BCMA or both antigens. In another embodiment, the targeted cells are malignant B cell lymphoma / leukemia such as, without limiting, B-ALL( B cell acute lymphoblastic leukemia) / B- LBL (B cell lymphoblastic lymphoma), high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.

[0292] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ncCAR (nanobody antibody CAR) polypeptide against cells expressing CD 19 or BCMA or both.

[0293] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ncCAR (nanobody antibody CAR) polypeptide to deplete autoantibody producing cells, such as plasma blasts or B cells associated with autoimmune diseases.

[0294] BCMA-CD19 ntCAR target cells expressing BCMA or CD 19 or both

[0295] In one embodiment, BCMA-CD19 nanobody antibody tandem CAR (“BCMA-CD 19 ntCAR”) has a dual domain to bind two different antigens within one dual-CAR. The structure of a dual domain is formed with VHH1-VHH2. BCMA-CD 19 ntCAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain( s) and CD3 zeta chain.

[0296] In one embodiment, BCMA-CD19 ntCAR has two particular antigen recognition sites (BCMA and CD19) are joined by a peptide linker, placed in tandem on a single intracellular domain and expressed as a single CAR on a cell surface. There are an irrespective order of BCMA and CD 19 selected.

[0297] In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ntCAR polypeptide against a cell expressing CD19 or BCMA or both.

[0298] In one embodiment, the present disclosure provides a method of treating leukemia or lymphoma or myeloma expressing CD19 or BCMA or both in a patient by administering a BCMA-CD19 ntCAR engineered cell to a patient in need thereof.

[0299] In further embodiments, the BCMA-CD19 ntCAR engineered cells target cells having cell surface CD 19 or BCMA or both antigens. In another embodiment, the targeted cells are malignant B cell lymphoma / leukemia such as, without limiting, B-ALL (B cell acute lymphoblastic leukemia) / B- LBL (B cell lymphoblastic lymphoma), high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.

[0300] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ntCAR (nanobody antibody CAR) polypeptide against cells expressing CD 19 or BCMA or both.

[0301] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ntCAR (nanobody antibody CAR) polypeptide to deplete autoantibody producing cells, such as plasma blasts or B cells associated with autoimmune diseases.

[0302] Humanized BCMA and CD 19 cCAR sequence

[0303] CARs traditionally are derived from murine monoclonal antibodies and have been shown to trigger antimurine immune responses in patients, increasing rates of rejection of CAR-T cell therapy. Humanization of the BCMA and CD 19 cCAR constructs is an effective way to bypass immune- mediated rejection of the therapy which can reduce rejection rates and increase the persistence of CAR-T cells in the patients. The humanized BCMA-CD19 structure may contain an anti-BCMA binding domain comprising of one or more of the three light chain complementary determining region and / or an anti-CD19 binding domain comprising of one or more of the three light chain complementary determining region,

[0304] In one embodiment, the disclosure provides a method of treating a cell proliferative disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a humanized BCMA-CD19 cCAR polypeptide against a cell expressing CD 19 or BCMA or both.

[0305] In one embodiment, the present disclosure provides a method of treating leukemia or lymphoma or myeloma expressing CD 19 or BCMA or both in a patient by administering a humanized BCMA- CD19 cCAR engineered cell to a patient in need thereof.

[0306] In further embodiments, the humanized BCMA-CD19 cCAR engineered cells target cells having cell surface CD 19 or BCMA or both antigens. In another embodiment, the targeted cells are malignant B cell lymphoma / leukemia such as, without limiting. B-ALL (B cell acute lymphoblastic leukemia) / B-LBL (B cell lymphoblastic lymphoma), high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.

[0307] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a humanized BCMA-CD19 ncCAR (nanobody antibody CAR) polypeptide against cells expressing CD 19 or BCMA or both.

[0308] In one embodiment, the disclosure provides a method of treating an autoimmune disease. The method includes administering to a patient in need thereof a therapeutically effective amount of an engineered cell expressing a BCMA-CD19 ncCAR (nanobody antibody CAR) polypeptide to deplete autoantibody producing cells, such as plasma blasts or B cells associated with autoimmune diseases. Methods of characterization of nanobody antibody CARs or humanized antibody CARs can be seen in the references (Li et al, Nat Commun. 2023 Sep 22; 14(1 ):5920; Chen et al. Leukemia. 2018 Feb; 32(2): 402-412).

[0309] Antibody ligands for treating cancers or autoimmune diseases

[0310] In one embodiment, the disclosure provides a composition for targeting of cancer cells, wherein the composition comprises one or more targeting ligands comprising an antibody fragment, wherein the antibody fragment comprises a VHH domain comprising a sequence or a variant or a derivative thereof. In some embodiments the one or more targeting ligands bind to one or more tumor types selected from the group of leukemia, lymphoma, myeloma, bladder carcinoma, breast carcinoma, cervical carcinoma, cholangiocarcinoma, colorectal carcinoma, gastric sarcoma, glioma, lung carcinoma, melanoma, osteosarcoma, ovarian carcinoma, pancreatic carcinoma, prostate carcinoma, stomach carcinoma, a head, a neck tumor, and a solid tumor.

[0311] In some embodiments the one or more targeting ligands bind to B cells or plasma cells, or both associated with autoimmune diseases.

[0312] In an embodiment, a recombinant polypeptide comprising one or more VHH antibodies.

[0313] In an embodiment, a multivalent antibody is provided comprising a first antigen-bind moiety comprising a VHH antibody and a second antibody binding moiety. In a further embodiment, the second antigen-binding moiety comprises a CD3 antibody binding to a T cell.

[0314] The multivalent antibody bears a VHH antibody comprising any CDR provided in table 1,2, 3, 4, 20, 21, 22 and 23.

[0315] In one embodiment the therapeutic agent is selected from the group consisting of a cytotoxin or radionuclide, or a chemotherapeutic agent. An object matter of the presently disclosed having been described above, the advantages of the presently disclosed subject matter will become apparent to those skilled in the art after a study of the following disclosed description and examples.

[0316] The CAR-T cell or ligand antibody described herein can be used to treat B-cell malignancies including but not limited to B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), Acute Lymphoblastic Leukemia (ALL), AIDS-related lymphoma, ALK- positive large B-cell lymphoma, Burkitt's lymphoma, Chronic Myelogenous Leukemia (CML), Chronic lymphocytic leukemia, (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma Classical Hodgkin lymphoma, Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma. Intravascular large B-cell lymphoma, Large B-cell lymphoma arising in HHV8-associated multicentric Castleman's disease, Lymphomatoid granulomatosis, multiple myeloma, myelodysplasia and myelodysplastic syndrome, Lymphoplasmacytic lymphoma, Mantle cell lymphoma (MCL), Marginal zone B-cell lymphoma (MZL), Mucosa- Associated, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions. Lymphatic Tissue lymphoma (MALT), Nodal marginal zone B cell lymphoma (NMZL), Nodular lymphocyte predominant Hodgkin's lymphoma, Non- Hodgkin's lymphoma, Plasmablastic lymphoma, Plasmacytoid dendritic cell neoplasm Primary central nervous system lymphoma, Primary effusion lymphoma, Splenic marginal zone lymphoma (SMZL), and Waldenstrom's macroglobulinemia. In some embodiments, the B-cell lymphoma is selected from the group consisting of Acute Lymphoblastic Leukemia (ALL), Chronic lymphocytic leukemia, (CLL), Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma, Mantle cell lymphoma (MCL), Marginal zone B-cell lymphoma (MZL), Mucosa-Associated Lymphatic Tissue lymphoma (MALT), and Non-Hodgkin's lymphoma. In some embodiments, the B -cell lymphoma is Non-Hodgkin' s lymphoma and "preleukemia" which are a collection of hematological conditions united by dysplasia of myeloid blood cells, and to disease associated with CD 19 expression include, but not limited to atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing CD 19, autoimmune conditions including but not limited to achalasia, Addison’s disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4-activating rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), diabetes mellitus, discoid lupus. Dressier’ s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis. Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s disease, Hashimoto’s thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), hypogammalglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4- related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis. Lichen planus, Lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, membranous nephropathy, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage- Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s syndrome, reactive Arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, sensitized / preformed antibodies in solid organ transplant, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener’s disease. Guillain-Barr syndrome, Hashimoto’s disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorders (MOGSD), neuromyelitis optica spectrum (NMOSD), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In preferred embodiments, the autoimmune disorder is not IgG4-related disease. In preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV), SLE (e.g. relapsed or refractory SLE), or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disorder is AAV (e.g. relapsed or refractory AAV) or rheumatoid arthritis (e.g. relapsed or refractory rheumatoid arthritis).

[0317] The CAR-T cell or ligand antibody described herein can be used to treat the cell proliferative disease selected from the group consisting of gastric cancers, breast cancers, colon cancers, liver cancers and pancreatic cancers. These antibody therapies or CAR-T cell therapies described herein may be used in combination with, preceding or following surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, stem cell or bone marrow transplant and / or hormone therapy.

[0318] Peptide

[0319] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein but all refer to a compound with two amino acids linked by a covalent peptide bond. A polypeptide is a compound that includes a minimum of two amino acids, with no maximum number, covalently linked by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0320] Antigen

[0321] An “antigen” may refer to any compound, substance, molecule or macromolecule including but not limited to proteins or polypeptides or even fragments of larger molecules, that has the potential to stimulate antibody production or a T cell response in humans and other animals alike, including compositions that may be comprise on or within cancer cells (tumor-specific proteins). An antigen may be absorbed, inhaled, ingested or injected into a human or animal. An antigen may react with the components or products of any component of the immune system including cellular immunity.

[0322] A “target antigen” may refer to an antigen of interest that is not found abundantly on the surface of nonual cells. A target antigen may be an antigen that can bind to the CAR’s binding domain.

[0323] An antigen may be expressed endogenously by genomic DNA or may be recombinantly expressed through genetic cloning methods. An antigen may be specific in nature, such as those expressed by cancer cells or on or in specific tissues, or it may express more broadly, even systemically. Some example of antigens may include but are not limited to 707-AP, AFP (alpha (a)-fetoprotein), ART-4, BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD 19, CD20,CD22, CD30, CD33, CD44v7 / 8 (cluster of differentiation 44, exons 7 / 8), CAMEL (CTL -recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide- 1), CASP-8 (caspase-8), CDC27m (cell-division cycle 27 mutated), CDK4 / m (cycline-dependent kinase 4 mutated), CEA (carcinoeinbryonic antigen), C-type lectin-like- 1 (CLL-1), CT (cancer / testis (antigen)), Cyp-B, DAM (differentiation antigen melanoma), EGFR, EGFRvlll, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP (folate binding protein), fAchR, G250 (glycoprotein 250), GAGE (G antigen), GD2, GD3, glypican 3 (GPC3), GnT-V (N-acetylglucosaminyltransferase V), GplOO (100-kD glycoprotein), HAGE, HER-2 / neu (EGFR2), HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL-13R-a2, KIAA0205, KDR, K-light chain, LAGE (L antigen), LDLR'FUT, LeY, LI CAM, MAGE (melanoma antigen), MAGE-A1, mesothelin, Murine CMV infected cells, MART-l / Melan-A, MCI R, Myosin / m (myosin mutated), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligands, NY-BR-1 (New York breast differentiation antigen 1 ), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), oncofetal antigen (h5T4), P15 (protein 15), pI90 minor bcr-abl (protein of 190KD bcr-abl), Pml / RARa (promyelocytic leukaemia / retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (pro state- specific antigen), PSCA (Prostate stem cell antigen), PS MA (prostatespecific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma XI , -2, -3, -4), TAA (tumor-associated antigen), TPL'm (triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein 1 , or gp75), TRP-2 (tyrosinase related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), or WT1 (Wilms' tumor gene).

[0324] Engineered cell

[0325] An "engineered cell” means any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells that contain the DNA or RNA sequences encoding a chimeric- antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on the cell surface. Isolated host cells and engineered cells may be used, for example, for enhancing an NK cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases.

[0326] In an embodiment, the engineered ceil includes immunoregulatory cells.

[0327] Antigen presenting cell

[0328] The term “Antigen presenting cell” or “APC” may refer to cells that are capable of processing antigens and presenting them to T cells. APCs include dendritic cells, macrophages, B cells, activated epithelial cells and other cells that possess the ability to stimulate the T- cell receptor (TCR) and / or are capable T cell stimulation.

[0329] Natural Killer (NK) cells are involved in the immune defense against viral infections and for the immunosurveillance of cancer cells and / or tumors. NK cells are involved in the recognition of non-healthy cells (either infected or cancerous) and in responses, release cytolytic granules such as cytokines and chemokines, e.g. IFN-y, leading to the death of the abnormal cell.

[0330] Macrophages are cells that play an important role in innate immunity where they recognize and phagocytose abnormal cells such as cancer cells, cellular debris, microbes or foreign substances that do not express molecules that are specific to healthy cells within the body. These fragmentized peptides from phagocytosed antigens are then presented on MHCII molecules on their cell surface to enable recognition by activated T helper cells.

[0331] In an embodiment, the engineered cell includes immunoregulatory cells,

[0332] Immunoregulatory cells include T-cells, such as CD4 T-ceils (Helper T-cells), CD8 T-cells (Cytotoxic T-cells, CTLs), and memory T cells or memory stem cell T cells. In another embodiment, T-cells include Natural Killer T-cells (NK T-cells), In an embodiment, the engineered cell includes Natural Killer ceils. Natural killer cells are well known in the art. In one embodiment; natural killer cells include cell lines, such as NK- 92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells.

[0333] NK cells mediate anti-tumor effects without the risk of GvHD and are short-lived relative to T- cells. Accordingly, NK cells would be exhausted shortly after destroying cancer cells, decreasing the need for an inducible suicide gene on CAR constructs that would ablate the modified cells.

[0334] In accordance with the present disclosure, it was surprisingly found that NK cells provide a readily available cell to be engineered to contain and express the chimeric antigen receptor polypeptides disclosed herein.

[0335] Allogeneic or autologous NK cells induce a rapid immune response but disappear relatively rapidly from the circulation due to their limited lifespan. Thus, applicants surprisingly discovered that there is reduced concern of persisting side effects using CAR cell-based therapy.

[0336] Polynucleotide

[0337] The term polynucleotide or nucleotide sequence refers to a sequence or chain of DNA or RNA comprised of nucleotide polymers. Nucleic acids and nucleotides may be used interchangeably. Nucleotides may be hydrolyzed into the basic monomeric units of nucleosides.

[0338] Polynucleotides may include all nucleotide sequences that are available and obtained through the means of but not limited to recombinant means i.e. genetic cloning of these sequences from a library or genome using standard and advanced cloning technology including but not limited to polymerase chain reaction (PCR) and other techniques. The polynucleotide may comprise of a gene, a part of a gene, or an expression or cloning cassette.

[0339] The polynucleotide may be cloned into a “vector” which may be comprised of an isolated polynucleotide and can be used to transport the polynucleotide to the interior of the cell. There are different types of vectors that may be common knowledge to anyone skilled in the art and may include linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, phagemid, cosmid, and viruses, including phages and phage derivatives.

[0340] Vector

[0341] A vector may also refer to an autonomously replicating virus or plasmid, non-plasmid and non- viral compounds that allow for transport of nucleotides into the cells including but not limited to polylysine compounds and liposomes.

[0342] The term vectors may also include but is not limited to cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors. Commonly used viral vectors include but are not limited to adenoviral vectors, adeno-associated virus vectors, alphaviruses, poxviruses, herpesviruses, retroviral vectors, lentiviral vectors, etc.

[0343] The term “viral based systems” or “viruses” may be used interchangeably to describe vector systems to facilitate gene transfer into mammalian cells. Engineered gene targets can be inserted into a vector and packaged into retroviruses that can then be used to deliver the described genes into patients through in vivo or ex vivo methods. Other viral categories to facilitate gene transfer include but are not limited to retroviruses, adenoviruses and lentiviruses. A vector contains an origin of replication that is functional in at least one organism, a promoter sequence, restriction endonuclease sites or one or more selectable marker.

[0344] Expression of chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to, at least one of a SFFV (spleen-focus forming virus) or human elongation factor I la (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor la (EF) promoter. Examples of less-strong / lower-expressing promoters utilized may include, but is not limited to, the simian vims 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or a part thereof. Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech Laboratories, Mountain View, CA) or a part or a combination thereof.

[0345] In a preferred embodiment, the promoter is an SFFV promoter or a derivative thereof. It has been unexpectedly discovered that SFFV promoter provides stronger expression and greater persistence in the transduced cells in accordance with the present disclosure.

[0346] To increase the safety profile of the CAR-T therapy, in one embodiment, the vector may contain an inducible suicide gene (‘‘safety switch”) or more than one safety switches that doesn’t interfere with the retroviral vector, lentiviral vector, adenoviral vector or plasmid. The "safety switch" may be an inducible suicide gene, such as, without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450.

[0347] In another embodiment the safety switch may be CD20 or CD 19 or truncated epidermal growth factor receptor in T cells to eliminated unwanted modified T cells.

[0348] In another embodiment, other possible safety switches have been contemplated.

[0349] Promoter

[0350] While retroviruses have been well accepted for their gene transfer capabilities, the efficient transfer of genes into human T cells is largely dependent on the internal promoter that drives gene expression. Third or fourth-generation CARs possess multiple co-stimulatory domains, and their large size may result in unequal gene expression necessitating the inclusion of a strong promoter. The promoter selected may be sourced to target different cell specificities and strengths. Because CAR-T cell therapies so heavily depend on the adequate expression of the CAR in vivo, the careful selection of an appropriate promoter is critical.

[0351] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. A CMV promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor - 1 a (EF- 1 a). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency vims (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the disclosure should not be limited to the use of constitutive promoters, inducible promoters are also contemplated as part of the disclosure. The use of an inducible promoter provides a molecular switch capable of turning on the expression of the polynucleotide sequence, which is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0352] Expression of chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to, at least one of a SFFV (spleen-focus forming virus) or human elongation factor I la (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor la (EF) promoter. Examples of less-strong / lower-expressing promoters utilized may include, but is not limited to, the simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or a part thereof. Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech Laboratories, Mountain View, CA) or a part or a combination thereof.

[0353] In a preferred embodiment, the promoter is an SFFV promoter or a derivative thereof. It has been unexpectedly discovered that SFFV promoter provides stronger expression and greater persistence in the transduced cells in accordance with the present disclosure.

[0354] The present invention includes the use of a strong promoter that drives high CAR gene expression in NIC and T cells. In one embodiment the promoter may be an SFFV promoter driving the expression of multiple modular units of CARs linked by a P2A peptide. In another embodiment, EF 1 a promoter may be used for cloning humanized scFv CAR sequences. In another embodiment, a pMXs retrovirus promoter contained within the 5’ LTR may be used to drive gene expression.

[0355] CAR structure

[0356] A CAR polypeptide consists of six domains: a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The chimeric nature of the receptor contains both antigen binding and T cell activating functions. Co-stimulatory domains are derived from various proteins and include but are not limited to CD28, CD2, 4-IBB (CD 137, also referred to as "4-BB"), and OX-40 (CD 124).

[0357] Compound CAR (cCAR) structure

[0358] A compound CAR consists of two CAR constructs linked by a self-cleaving peptide. The first CAR consists of a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The first CAR is followed by a self-cleaving peptide that serves to link the two independent units of CARs, together during expression. The second CAR contains a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The antigen recognition domains are selected from the group of BCMA, CD 19, CD20, CLL1, CD33 and CD7.

[0359] A compound CAR consists of two CAR constructs linked by a self-cleaving peptide. The first CAR consists of a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The first CAR is followed by a self-cleaving peptide that serves to link the two independent units of CARs, together during expression. The second CAR contains a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The antigen recognition domains, irrespective of order are selected from the group consisting of BCMA, CD20, CLL1, CD33 CD19 and CD7.

[0360] A compound CAR consists of two CAR constructs linked by a self-cleaving peptide. The first CAR consists of a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The first CAR is followed by a self-cleaving peptide that serves to link the two independent units of CARs, together during expression. The second CAR contains a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain. The antigen recognition domains, irrespective of order are selected from the groups consisting of BCMA and CD19. CLL1 and CD33, CD19 and CD7, or BCMA and CD7.

[0361] The compound CAR can therefore target the same population of tumor cells or two or more different populations of tumor populations. For example, the first CAR-T cell may target a tumor population and the second CAR-T cell may target cancer or leukemic stem cells to prevent cancer relapse.

[0362] The compound CAR can therefore target the same population of immune cells or two or more different populations of immune cell populations. For example, the first CAR-T cell may target a B cell population and the second CAR-T cell may target a plasma cell population.

[0363] In one embodiment, the compound CAR-T cell may target different or the same tumor populations to combat tumor factors causing cancer cells resistant to the CAR-T cell’s killing activity therefore down regulating the target antigen and preventing antigen escape.

[0364] Signal peptide

[0365] A "signal peptide" is defined as a peptide sequence is responsible in directing the transport as well as the localization of the peptide and any attached polypeptide within a cell to a region within the cell (e.g. to a certain cell organelle like the endoplasmic reticulum) and / or the surface of the cell. The signal peptide is a peptide that may be secreted, or it may be a transmembrane protein involved in directing the polypeptide of this disclosure to the cell membrane and the cell surface. The signal peptide may be involved in the correct localization of the polypeptide of disclosure. The signal peptide of this disclosure directs the polypeptide of this disclosure to the cell membrane where the extracellular domain of the polypeptide is displayed on the surface of the cell, the transmembrane domain spans the plasma membrane of the cell, and the activation domain is within the interior of the cell i.e. the cytoplasm. The signal peptide may be cleaved after passage through the endoplasmic reticulum and may be referred to as a cleavable signal peptide. The signal peptide may be a human protein of type 1, II, III, or IV. The signal peptide may include an immunoglobulin heavy chain signal peptide. Some commonly used signal peptides include but are not limited to Secrecon, Human IgK VIII, Human IgG V, tPA, Consensus and mouse IgK VIII.

[0366] Antigen recognition domain

[0367] The "antigen recognition domain" consists of a polypeptide that is specific to the recognition of the target antigen, receptor, peptide ligand, or protein ligand or a target polypeptide. The antigen recognition domain can be obtained from various extracellular domains or secreted proteins associated with ligand binding and / or signal transduction. The antigen recognition domain comprises of a portion of Ig heavy chain linked with a portion of Ig light chain, together forming a single chain fragment variable (scFv) that binds specifically to a target antigen. The antibody nature may be monoclonal, polyclonal or of any type that binds specifically to the target antigen. The antigen recognition domain may be a receptor or ligand, or it may be specific for a specific disease condition of any kind so long as the disease condition has a cell surface antigen that may be recognized by at least one of the chimeric receptor constructs present in the compound CAR architecture. The chimeric receptor may be for any cancer for which a specific monoclonal or polyclonal antibody exists or is capable of being generated. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia have antigens specific for the chimeric receptors.

[0368] The antigen recognition domain may consist of an antigen binding domain that may be derived from an antibody against an antigen of the target, or a peptide binding an antigen of the target, or a peptide or protein binding an antibody that binds an antigen of the target, or a peptide or protein ligand (including but not limited to a growth factor, a cytokine, or a hormone) binding a receptor on the target, or a domain derived from a receptor (including but not limited to a growth factor receptor, a cytokine receptor or a hormone receptor) binding a peptide or protein- ligand on the target. The antigen recognition domain also consists of the binding moiety or variable region of a monoclonal or polyclonal antibody that is selective for the target.

[0369] In one embodiment, the antigen recognition domain may consist of a scFv or VHH that specifically binds to CD19. In one embodiment, the antigen recognition domain may consist of a scFv or VHH that specifically binds to BCMA. In one embodiment, two or more antigen recognition domains may consist of a scFv or VHH that specifically binds to CD19.

[0370] In one embodiment, the antigen recognition domain may consist of a VHH that specifically binds to CD 19. In one embodiment, the antigen recognition domain may consist of a VHH that specifically binds to BCMA. In one embodiment, two or more antigen recognition domains may consist of a VHH that specifically binds to CD 19.

[0371] In one embodiment, the antigen recognition domain may consist of a VHH that specifically binds to C laudin 18.2.

[0372] In another embodiment, the antigen recognition domain may consist of a sequence that possesses some variability but may still be selective for its targets. The polypeptide of the antigen recognition domain may be at least 95%, at least 90%, at least 80%, or at least 70% identical to the antigen recognition domain polypeptide disclosed herein and still be selective for the targets described herein and still be within the scope of this disclosure.

[0373] Hinge region

[0374] The “hinge region” or a “spacer region” is a peptide sequence positioned between for example, including, but not limited to, the chimeric antigen receptor, and at least one co-stimulatory domain and a signaling domain. The hinge polypeptide sequence may be obtained from any suitable sequence from any genus including human, or a part thereof. A CAR may contain one or more hinge domains following the binding region of the CAR. The preliminary role of the hinge domain is to position the antigen binding domain away from the effector cell in order to enable cell / cell contact to promotes antigen binding and activation. The hinge domain may be derived from many sources include natural, synthetic, semi-synthetic, recombinant or any other source that may be known to one skilled in the art. The hinge domain may include the peptide sequence that is naturally occurring or genetically engineered.

[0375] Commonly used hinge regions may include but are not limited to those derived from or comprise of fragments of any immunoglobulin-like hinge domain including but not limited to IgGl, IgG2, IgG3, lgG4, IgA, IgD, IgE, or IgM, CD8a, CD4, CD28 and CD7, CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8oc, CD8p, CD1 la (ITGAL), CD1 lb (ITGAM), CD1 1c (ITGAX), CD1 Id (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SEAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CFACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CEEC2), CD79A (B-cell antigen receptor complex- associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD 100 (SEMA4D), CD 103 (ITGAE), CD 134 (0X40), CD 137 (4- 1BB), CD150 (SLAMF1), CD158A (K1R2DL1), CD158B1 (KJR2DL2), CD158B2 (K1R2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-l), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), inducible T cell co-stimulator (ICOS), LFA-1 (CD1 la / CD18), NKG2C, DAP- 10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule. MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, or Toll ligand receptor, or which is a fragment or combination thereof.

[0376] Transmembrane domain

[0377] The “transmembrane domain” is a hydrophobic polypeptide that spans the entire cellular membrane from the outside of the cell (extracellular) through the plasma membrane of the cell to the inside of the cell (intercellular or cytoplasmic). The transmembrane domain may be structured as an alpha helical domain or a beta-barrel domain or a combination thereof. The transmembrane domain may include a polytopic protein consisting of alpha-helices, beta sheets or fragments and combinations thereof.

[0378] The transmembrane domain may contain a polytopic protein that consists of several transmembrane segments each structured as alpha-helical, beta sheets, or combinations thereof.

[0379] In one embodiment, the transmembrane domain is naturally associated with one of the domains in the CAR. In another embodiment, the transmembrane domain contains amino acid substitution to avoid binding of this transmembrane domain to other transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0380] Commonly used transmembrane domains include but are not limited to those derived from alpha, beta or zeta chain of the T-cell receptor, a CD3 zeta chain, CD3 epsilon. CD28, CD3e., CD45, CD4, CD5, CD8, CDS, CDS, CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD 134, CD 137, ICOS, CD 154. GITR

[0381] Signaling domain

[0382] The “signaling domain” and “co-stimulatory domain” comprise polypeptides that modulate the immune activate of the cell to activate or stimulate at least some aspect of the cell’s signaling pathway. It is a functional protein that transmits information within the cell to regulate cellular activity by generating secondary messengers or responding to such messengers as an effector

[0383] Commonly used costimulatory domains include those derived from 4-1BB, CD2, CD 3 delta, CD 3 epsilon, CD 3 gamma, CD 3 zeta, CD 4, CD7, CD8a, CD8p, CD8q, CD 11 a (ITGAL), CD1 lb (IT GAM), GDI 1c (ITGAX), CD1 Id (IT GAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGAl), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1 ), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD 79 A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex- associated beta chain), CD84 (SLAMF5),CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (0X40), CD137 (4- IBB), CD150 (SLAMFl), CD158A (KTR2DL1), CD158B 1 (KIR2DL2), CD158B2 (KTR2DL3),CD158C (KTR3DP 1), CD158D (KIRDL4), CD158F 1 (KTR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD 160 (BY55), CD 162 (SELPLG), CD226 (DNAM1), CD229(SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF 14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 ( K-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRT AM), CD357 (TNFRSF18), GITR, inducible T cell costimulatory (ICOS), 1COS-1, LFA-1 (CD 1 la / CD18), KG2C, DAP- 10, ICAM- 1, NKp80 (KLRF 1), OX-40, IL-2R beta, IL-2R gamma, IL-7R alpha, LFA- 1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, a CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, a TNF receptor protein, an immunoglobulin protein, a cytokine receptor, an integrin, activating NK cell receptors, a Toll ligand receptor, and fragments or combinations thereof CD.

[0384] EXAMPLES

[0385] In some embodiments, camels were immunized, and a phage display nanobody library was constructed for screening as a result of which cam-BCMA VHH antibody (Also called BCMA VHH) against BCMA was obtained (Figure IA) and described in Table 1 and Table 2.

[0386] Table 1: FR and CDR amino acid sequences of a BCMA VHH nanobody antibody (also called

[0387] BC4)

[0388] Table 2: FR and CDR amino acid sequences of a BCMA VHH nanobody antibody (also called BC5)

[0389] In some embodiments, camels were immunized, and a phage display nanobody library was constructed for screening as a result of which cam-CD 19 VHH antibody (Also called CD 19 VHH ) against CD19 was obtained (Figure 1A) and described in Table 3 and Table 4.

[0390] Table 3: FR and CDR amino acid sequences of a CD 19 VHH nanobody antibody (also called BS040-2)

[0391] Table 4: FR and CDR amino acid sequences of a CD 19 VHH nanobody (also called BS040-10) In one embodiment, the engineered cell with a GDI 9 nanobody antibody CAR (CD 19 nCAR) comprises CD 19 VHH nanobody antigen recognition domain (binding domain), SEQ ID NO. 24 and 32.

[0392] In one embodiment, the engineered cell with a BCMA nanobody antibody CAR (BCMA nCAR) comprises BCMA VHH nanobody antigen recognition domain (binding domain), SEQ ID NO. 8 and 16.

[0393] In one embodiment, the engineered cell with a Ciaudin 18.2 nanobody antibody CAR (Claudin 18.2 nCAR) comprises Claudin 18.2 VHH nanobody antigen recognition domain (binding domain), SEQ ID NO. 40.

[0394] In one embodiment, the engineered cell with a CLLlnanobody antibody CAR (CLL1 nCAR) comprises CLL1 VHH nanobody antigen recognition domain (binding domain), SEQ ID NO. 112, 120 and 128.

[0395] In one embodiment, the camelid antibody gene sequence was synthesized into the pTT5-hIgGl Fc (LALA) expression vector and purified using SEC-HPLC. The antibody expression format is VnH-human IgGl Fc (LALA).

[0396] In one embodiment, 5 pairs of humanized CD19 antibodies were designed. In one embodiment, 4 pairs of humanized BCMA antibodies were designed.

[0397] In one embodiment, the humanized CDR constructs for CD 19 was determined using deep learning as described in Table 3 and Table 4. Different CDRs were generated for each heavy and light chain to test for binding affinity of each humanized construct in comparison with the wildtype CD 19 derived from mouse.

[0398] In one embodiment, the humanized antibody gene sequence synthesized into the pTT5-hlgGl Fc (LALA) expression vector. In another embodiment, the humanized antibody contains a heavy chain and a light chain as described in Table 16.

[0399] In another embodiment, the humanized antibody contains a heavy chain and a light chain amino acid sequence as described in Table 24 and Table 25.

[0400] The antibody expression format is scFv -human IgGl Fc (LATA).

[0401] Target Protein Expression

[0402] VHH antibody was isolated using the same strategy described in Figure 1A. In one embodiment, the protein expression was carried out in HEK293 cells. In another embodiment, the protein expression was carried out in CHO cells. The expressing vector was created to express VHH fused to human IgGl FC.

[0403] In another embodiment, cells were transfected at a density of 3x 106 / mL. PEImax and VHH-IgGl construct DNA were diluted in culture media to allow for the formation of the PEI max-DNA. This complex was then added dropwise to the cells which were then cultured in a CO2 incubator. 24 hours following transfection OPM-293 ProFeed protein-free feed and anti-aggregation agent were added to the cell culture media. On the 5thday after transfection the cells were collected for the analysis of protein expression.

[0404] In one embodiment, protein expression was carried out through transient expression in HEK293 cells.

[0405] Antibody purification for BC4 and BC5 against BCMA antigen

[0406] In one embodiment SDS-PAGE was performed and proteins were separated by size to verify the identity of the expressed proteins.

[0407] In another embodiment, size exclusion chromatography-based high-performance liquid chromatography (SEC-HPLC) was carried out to verify the identity of the expressed proteins. In one embodiment, the purity of BC4 was determined using SEC-HPLC as described in Figure IB.

[0408] In another embodiment, the purity of BC5 was determined using SEC-HPLC as described in Figure 1C.

[0409] BC4 and BC5 VHH antibody binding activities

[0410] To test the specificity of the purified antibodies to BCMA, an ELISA was carried out between the purified antibodies and other proteins that are expressed on the cell surface of B cells namely. Transmembrane activator and CAME interactor (TACI) and B-cell activating factor receptor (BAFFR) as depicted graphically in Figure 2A and Figure 2B and Figure 2C. As expected, no binding between BC4 / BC5 and either TACI or BAFFR was observed, when compared to a BCMA positive control and a negative isotype control (hlgGl). EC50-hBCMA using ELISA test was determined in Table 5

[0411] Table 5: ELISA protein binding results for BC4 and BC5 to human BCM A

[0412] Table 6: FACS protein binding results for CHO KI cells expressing BCMA.

[0413] Following the validation of the binding affinity and specificity of BC4 and BC5 VHH antibodies to BCMA, the ability of these antibodies to bind BCMA specifically on the cell surface was yet to be determined. To validate this specific binding affinity, the purified BC4 and BC5 VHH antibodies were co-incubated with CHO KI cells expressing BCMA on their cell surface and the binding affinity was measured using flow cytometry (FACS) as depicted graphically in Figure 3, the EC50 of which is calculated and tabulated in Table 6.

[0414] Table 7: Affinity test results of hBCMA and VHH-IgGl Fc

[0415] Finally, a BLI assay was conducted to screen the camelid BC4 and BC5 VHH proteins. BLI works by measuring the interference pattern obtained by reflected white light from a biomolecule layer that is immobilized on a fixed surface such as the tip of the sensor. This allows for real-time detection of protein interaction in solution. The results of the BLI assay are described in Figures 4. The association and dissociation kinetics are represented in table 7, where ka / kd is Kon / Koff .

[0416] Antibody purification for two CD19 VHH (BS040-2) and CD19 VHH ( BS040-10 ) against CD19 antigen

[0417] Using the same approach as described above related to the isolation and identification of two CD 19 VHH nanobody antibodies, CD19 VHH-IgGl constructs were generated to express their proteins.

[0418] In one embodiment, the purity of BS040-2 was determined using SEC-HPLC as described in Figure 4A.

[0419] In another embodiment, the purity of BS040-10 was determined using SEC-HPLC as described in Figure 4B.

[0420] CD 19 VHH antibody binding activities

[0421] In one embodiment the purified camelid BCMA VHH antibody (BS040-2) constructs were screened for their ability to bind human CD 19 His-tag.

[0422] In one embodiment the purified camelid CD 19 VHH antibody (BS040-10) constructs were screened for their ability to bind human CD 19 His-tag. In another embodiment, the binding affinity of the camelid CD 19 VHH antibodies was determined by ELISA as described in Table 8 and Figure 5. Tafasitamab is a humanized monoclonal antibody targeting CD 19 used as a positive control.

[0423] Further screening was conducted to measure the binding affinity of the camelid CD19 VHH antibodies purified through SEC-HLPC to Raji cells expressing CD19, engineered HEK293T cells expressing CD19 (huCD19-HEK293T) and Nalm6-Luc 2 cells expressing CD19. Tafasitamab was utilized as a positive control binding agent. The results were quantified by flow cytometry and described in Tables 9, 10 and 11 and plotted in Figures 6, 7 and 8.

[0424] Table 9: FACS protein binding results for Raji cells

[0425] Table 10: FACS protein binding results for HEK293T cells expressing CD 19

[0426] Table 11 : FACS protein binding results for Nalm6-Luc2 cells

[0427] Finally, a BLI assay was conducted to screen the camelid CD 19 VnH proteins with a BLI test. BLI works by measuring the interference pattern obtained by reflected white light from a biomolecule layer that is immobilized on a fixed surface such as the tip of the sensor. This allows for real-time detection of protein interaction in solution. The results of the BLI assay are described in Figure 9. The association and dissociation kinetics are represented in table 12,

[0428] Table 12: Binding kinetics of cam-CD19 VHH proteins to human CD19 His-tag protein

[0429] Target Protein Expression for Claudin 18.2 VHH and its bindings

[0430] Using the same approach as described above related to the isolation and identification of Claudin 18.2 VHH nanobody antibody Table 20) and its constructs were generated to express their proteins.

[0431] In some embodiments, camels were immunized, and a phage display nanobody library was constructed for screening as a result of which cam-Claudin 18.2 VHH nanobody antibody (Also called BS006-111 or Claudin 18.2 VHH) against Claudin 18.2 was obtained described in Table 20.

[0432] In one embodiment, the purity of Claudin 18.2 VHH was determined using SEC-HPLC as described in Figure 10. In one embodiment, the binding affinity of the purified Claudin 18.2 nanobody to HEK293 T-cells expressing Claudin 18.2 is described in Figure 11.

[0433] The specificity of Claudin 18.2 nanobody antibody is determined in the Figure 11 and 12. The result shows that Claudin 18.2 nanobody antibody (BS006-111) bind specifically to HEK293T- cells expressing Claudin 18.2 but not to HEK293T-cells expressing Claudin 18.1.

[0434] Following the validation of the purity of the purified Claudin 18.2 VHH antibody, it was tested using FACS, with antibody concentrations ranging from 0.01 nM to 1000 nM. Human IgGl was used as a negative control (isotype control) and Claudin 18.2 VHH hFc was used as a positive control. The EC50 measured the half maximal effective concentration and was calculated as depicted in Table 13

[0435] Table 13: Calculated EC50 values obtained from FACS between purified antibodies and Claudin 18.2 nanobody.

[0436] To further test the binding specificity of the Claudin 18.2 VHH antibody, the proteins were HEK293T cells expressing Claudin 18.1. Anti-Claudin 18.1 VHH hFc (VHH hFc) was used as a positive control. An isotype control was used as a negative control. These results validated that the isolated Claudin 18.2 VHH antibody (also called BS0061-111 ) selectively binds to Claudin 18.2 (Figure 1 1) and not Claudin 18.1 (Figure 12).

[0437] Humanized CD 19 and BCM A scFv

[0438] CDR-grafting involves analyzing the degree of overall and key structural amino acid matching using sequence similarity comparison. The job line template is selected for transplantation based on internal expression, druggability and other data. The best template sequence is selected for humanization. The mouse antibody CDR is transplanted into the selected germline and CDR determination methods are used to construct different humanized construct using machine learning and deep learning. Modeling and analysis of the mouse derived antibodies allows for identification of the FR (framework) amino acids of CDR introductions, allows for reverse mutations to be performed and for the final design of the basic humanized antibodies. The final construct is analyzed for its structured, post-translational modifications (PTM), immunogenicity and drugability of the humanized antibodies to determine and fine-tune the final constructs.

[0439] In some embodiments, CDR-grafting was utilized to generate humanized BCMA CAR constructs.

[0440] In some embodiments, CDR-grafting was utilized to generate humanized CD 19 CAR constructs.

[0441] In one embodiment, 5 pairs of humanized CD 19 antibody sequences were generated as described in Table 14 for further screening their bindings.

[0442] Table 14: Humanized CD 19 amino acid sequences

[0443] In one embodiment, 4 pairs of humanized BCMA antibody sequences were generated as described in Table 15 for further screening their bindings.

[0444] Table 15: Humanized BCMA amino acid sequences

[0445] In one embodiment, the humanized CD19 antibody gene sequence synthesized into the pTT5- hlgGl Fc (LALA) expression vector (Table 16).

[0446] Table 16: Expression information of humanized CD 19 scFv antibodies

[0447] Many versions of humanized

[0448] CD 19 scFv, CD19-hzl,

[0449] CD19-hz2, CD19-hz3 and CD19-hz4 were generated and expressed, purified using SPE-HPLC.

[0450] The purified proteins were then tested to their binding to human CD 19 His-tag protein measured by ELISA. The results of the ELISA suggested that CD19-hzl and CD19-hz2 bind the most efficiently to human CD19 His-tag protein. The humanized CD 19 antibody constructs are ranked by the affinity by which they bind the humanized CD 19 in Table 17 and Figure 13B.

[0451] Table 17: EC50-ELISA

[0452] In another embodiment, the different purified humanized BCMA scFv constructs were screened for their ability to bind human BCMA His protein.

[0453] In another embodiment, the binding affinity of the humanized BCMA scFv antibodies were determined by ELISA as described in Table 18 and Figure 13 A.

[0454] Table 18: ELISA protein binding results

[0455] The final EC-50 value of the ability of each humanized BCMA antibody construct to bind Human- BCMA His was determined and is listed in Table 19.

[0456] Table 19: EC50-ELISA

[0457] Further screening was conducted to measure the binding affinity of the humanized BCMA ScFv antibodies purified through SEC-HLPC to RPMI8226 celline expressing BCMA and HEK293 cells (negative for BCMA). Here, the humanized BCMA hz-1 and BCMA hz-3 antibody scFv antibodies displayed the highest binding to RPMI8226 cells as detected by flow cytometry and is described in Figure 14A. Similarly, the binding affinity of the humanized BCMA constructs to HEK293 cells was also determined by flow cytometry and quantified in Figure 15 A. These results indicate that the BCMA antibody clones displayed the binding to HEK293 cells close to that of the isotype control as detected by flow cytometry.

[0458] Further screening was conducted to measure the binding affinity of the humanized CD19 antibodies purified through SEC-HLPC to Raji expressing CD 19 and HEK293 cells (negative for BCMA). Here, the humanized BCMA hz-1 and BCMA hz-3 antibody clones displayed the highest binding to Raji cells as detected by flow cytometry and is described in Figure 14B. Similarly, the binding affinity of the humanized CD 19 constructs to HEK293 cells was also determined by flow cytometry and quantified in Figure 15B. These results indicate that the CD19 antibody clones displayed the binding to HEK293 cells close to that of the isotype control as detected by flow cytometry. Table 20: FR and CDR amino acid sequences of a Claudin 18.2 VHH nanobody antibody

[0459] CAR armored with secreted IL-15 / IL-15sushi

[0460] The combination of a CAR and sushi domain of the IL-15 alpha receptor (called IL-15sushi) is assembled on an expression vector and their expression is driven by a promoter. CAR with IL- 15 / IL-l 5 sushi is linked with the P2A self-cleaving sequence (Figure 16Aand 16B). CAR and IL-15 / IL- 15 sushi can be present on the T or NK cells.

[0461] To test CD19-IL-15 / IL-15sushi CAR function in vivo, we established xenogeneic mouse models. Mice were injected with Reh tumor cells (0.5x106cells / mouse) expressing luciferase on Day 1. On Day 3, IVIS was conducted to assay the appearance of REH cells. On Day 4, CD 19 CAR, and CD19-IL15 / IL15sushi CAR T-cells (CD19 CAR T cells expressing lL-15 / IL15sushi) were injected (~7.5x106total cells / mouse) and on day 6 through 32, IVIS imaging was conducted to assay semi-quantitative assessment of tumor burden and subsequent tumor depletion and control of tumor growth. After day 32, all CD 19 CAR T cell mice showed signs of severe tumor relapse, while CD19-IL- 15 / IL15sushi CAR T treated mice showed no sign of tumor (Figure 17). As time passed, REH tumor relapsed in standard CAR T treatment (CD 19 CAR T cells); however, the armored CAR persisted and depleted relapsed tumor, keeping mice disease free.

[0462] Example for BC4-Q-VAC nCAR (also called BC4-O-VAC CAR)

[0463] The combination of a BC4-Q-VAC nCAR and IL- 15 fused to the sushi domain of the IL- 15 alpha receptor (also called VAC) is assembled on an expression vector and their expression is driven by a promoter shown in Figure 18A. BC4-Q-VAC CAR consistent of a nanobody antibody VHH BCMA nanobody antibody (also called BC4), CD8 leader sequence, CD8 hinge and transmembrane regions, and a CD28 co-activator fused to the CD3zeta signaling domain (Figure 18A). The hinge region also contains two RTX-binding epitopes (also called Q) for anti- CD20 binding as a safety switch.

[0464] Transduced T cells efficiently express BC4-Q-VAC CARs -Activated human peripheral blood T cells were transduced with the retroviral virtues expressing BC4-Q-VAC CAR (also called BC4 nCAR VAC). Figure 18A shows the transduction efficiency between activated T cells transduced with either control vector or BC4-Q-VAC CAR construct, as determined by labeling with goat- VHH antibody and streptavidin-PE conjugate. Activated T cells transduced with the CAR viruses resulted in 64.25% VHH positive cells for BC4-Q-VAC nCAR.

[0465] BC4-Q-VAC nCAR T cells exhibit significant anti-tumor activity and greater persistence than control T cells in xenogeneic mouse model - hr order to evaluate the specific in vivo anti-tumor activity of BC4-Q-VAC CAR T cells against human tumor cell lines, we developed a xenogeneic mouse model using NSG mice sublethally irradiated and intravenously injected with 1 x 10e6 of luciferase-expressing U937-BCMA, acute myeloid leukemia cell line cells, which express BCM A on the cell surface, to induce measurable tumor formation (Figure 18C). Six days following tumor cell injection, all mice were intravenously injected with a course of a low dose, - 10 x 10Λ6 of either control T cells or BC4-Q-VAC nCAR T cells. On Day 6 (the day before CAR T cell treatment), Day 8 (48 hours after T cell treatment), and periodically thereafter, mice were subjected to IVIS imaging to measure leukemic killing. Average light intensity measured for the U937-BCMA mice injected with BC4-Q-VAC nCAR T cells was compared to that of mice injected with the control T cells to determine percent lysis of targeted cells. Results showed that only 3 days following treatment with CAR T cells (Day 8), mice treated with either CAR T cells had significantly lower tumor burden than mice given control T cells. By Day 18, BC4-Q- VAC nCAR almost completely eliminated leukemic cells as compared to that of control mice. (Figure 18C and 18D). The Kaplan-Meier survival analysis showed that mice treated with BC4- Q-VAC CAR T cells had significantly prolonged survival compared to mice treated with control T cells (p==0.0031) (Figure 18E). These results demonstrate a remarkable efficacy and long-term effects of BC4-Q-VAC nCAR T cells against a BCMA-positive tumor cell line in vivo.

[0466] Example for BC5-0-VAC nC AR

[0467] BC5-Q-VAC CAR consistent of a nanobody antibody VHH BCMA nanobody antibody (also called BC5), CD8 leader sequence, CD8 hinge and transmembrane regions, and a CD28 co- activator fused to the CD3zeta signaling domain (Figure 19A). The hinge region also contains two RTX-binding epitopes (also called Q) for anti-CD20 binding as a safety switch.

[0468] Transduced T cells efficiently express BC5-Q-VAC nCAR (also called BC5-Q-VAC CAR) - A schematic showing a BC5-Q-VAC CAR equipped with a cytokine complex, IL-15 / lL-15sushi, and a safety switch. Human peripheral blood T cells were activated and transduced with the retroviral vector containing the BC5-Q-VAC nCAR (Figure 19B). The analysis showed that 50.93% of the transduced T cells were VHH positive for BC5-Q-VAC nCAR.

[0469] BC5-Q-VAC CAR T cells exhibited robust anti-tumor activity and greater persistence than control T cells in a xenogeneic mouse model - The specific in vivo anti-tumor activity of BC5-Q- VAC nCAR T cells was evaluated using a xenogeneic mouse model. NSG mice were sublethally irradiated and injected with 1 x 10e6 luciferase-expressing U937-BCMA, an acute myeloid leukemia cell line, which express BCMA on the cell surface. Six days post-tumor cell injection, the mice were treated with a low dose (10 x 10e6) of either control T cells or BC5-Q-VAC nCAR T cells. On Day 5 (the day before T cell treatment), Day 8 (48 hours after T cell treatment), and periodically thereafter, the mice were subjected to IVIS imaging to measure leukemic killing (Figure 19C). The average light intensity measured for the U937-BCMA mice treated with BC5-Q-VAC CAR T cells was compared to that of mice injected with control T cells to determine percent lysis of targeted cells. Results showed that by Day 8, mice treated with BC5-Q-VAC CAR T cells had significantly lower tumor burden compared to control T cells (Figure 19C). By Day 18, BC5-Q-VAC nCAR T cells showed near complete elimination of leukemia cells as compared to the control mice. (Figure 19D). The Kaplan-Meier survival analysis indicated that mice treated with BC5-Q-VAC CAR T cells had significantly prolonged survival compared to those treated with control T cells (p=0.0031 ) (Figure 19E). These findings demonstrate the unexpected efficacy and long-term effects of BC5-Q-VAC nCAR T cells in targeting BCMA-positive leukemic cells in vivo.

[0470] In particular embodiments, the engineered cell includes BCMA nCAR linked to IL15 / IL-15sushi via the P2A cleavage sequence. A polypeptide providing this embodiment includes SEQ ID No. 61, 64 and corresponding polynucleotide sequence SEQ ID No. 60, 63.

[0471] In an embodiment, the engineered cell includes BCMA nCAR having a polypeptide providing this embodiment includes SEQ ID No. 86, 88 and corresponding polynucleotide sequence SEQ ID No. 85, 87.

[0472] Example for AB19-N2-28-I5R nCAR (anti-CD19 nanobodv CAR armored with IL- 15 / IL 15 sushi)

[0473] AB19-N2-28-15R nCAR consistent of a nanobody antibody VHH CD19 nanobody antibody (also called N2-15R), CD8 leader sequence, CD8 hinge and transmembrane regions, and a CD28 co-activator fused to the CD3zeta signaling domain (Figure 20A). Transduced T cells efficiently express AB19-N2-28-15R nCARs - A schematic show'ing the AB19-N2-28-15R nCAR equipped with a cytokine complex, IL-15 / IL-15sushi (also called IL15R), and a self-cleaving sequence. Human peripheral blood T ceils were activated and transduced with the retroviruses containing the AB19-N2-28-15R nCAR (Figure 20B) showing 83.32% CAR expression.

[0474] AB19-N2-28-15R nCAR T cells effectively lyse target REH cells expressing CD 19 in vitro - The cytotoxic potential of AB19-N2-28-15R nCAR T cells was assessed by co-culturing them with REH tumor cells expressing CD 19 antigen. The assays involved co-culturing either control T cells or AB19-N2-28-15R nCAR T cells with REH cells at effector to target (E:T) ratios of 0.1:1, 0.5: 1, and 1 :1 for 8 hours. Post-incubation, cells were stained with mouse anti-human CD3 and CD 19 antibodies and analyzed by flow cytometry. The results demonstrated that AB19-N2-28- 15R nCAR T cells completely lysed the target cells at all tested ratios, confirming their remarkable potent cytotoxic capability (Figure 20C).

[0475] Example for AB19-N2-BB nCARs

[0476] AB19-N2-BB- nCAR consistent of a nanobody antibody VHH CD 19 nanobody antibody (also called N2), CD8 leader sequence, CD8 hinge and transmembrane regions, and a 4- IBB co- activator fused to the CD3zeta signaling domain (Figure 21 A). Transduced T cells efficiently express AB19-N2-BB nCARs.

[0477] Expression of AB19-N2-BB nCAR - Expression of the AB19-N2-BB nCAR was measured by FACS (Figure 21B) in donor T cells transduced with AB19-N2-BB nCAR retroviruses. FACS analysis showed that AB 19-N2-BB nCAR was able to be expressed on roughly 84.66% of the T cells.

[0478] Co-culture killing assays - Co-culture killing assays, in which target tumor cell lines expressing the CD 19 was incubated with AB19-N2-BB nCAR or mock T cells, were employed to determine the anti-tumor function of AB 19-N2-BB nCAR T cells in vitro against the CD19+ cell line. Co- culture experiments were performed at effector to target (E:T) ratios of 0.1 : 1 , 0.5 : 1 , and 1 : 1 for 8 hours and were then directly analyzed by flow cytometry with mouse anti-human CD3 PerCP and mouse anti-human CD19-APC. Each assay was consisted of target cells (REH cells expressing CD19) incubated with either mock control or CAR T-cells (Figure 21C). REH is an acute lymphoblastic leukemia cell line. This experiment revealed the dose-dependent nature of the AB19-N2-BB nCAR T and even at very low E:T ratios such as 0.1 : 1, there is near complete lysis of tumor cells. At the 1 : 1 ratio, killing ability was observed with virtually all tumor cells lysed.

[0479] In particular embodiments, the engineered cell includes CD19 nCAR linked to IL15 / IL-15sushi via the P2A cleavage sequence. A polypeptide providing this embodiment includes SEQ ID No.

[0480] 66 and corresponding polynucleotide sequence SEQ ID No. 65. In an embodiment, the engineered cell includes CD 19 nCAR having a polypeptide providing this embodiment includes SEQ ID No. 68, 76, 78 and corresponding polynucleotide sequence SEQ ID NOS. 67, 75, 77.

[0481] Example for BC4-AB19-N2-15R ncCAR

[0482] BC4-AB19-N2-15R ncCAR (also called BCMA-CD19-IL-15 / IL15sushi ncCAR or ncCAR) construct bears complete two units of CARs (Figure 22A). Each consists of the leader sequence, VHH nanoantibody binding domain, hinge region, transmembrane domain, co-stimulatory domain and CD3 zeta chain signaling domain. An enhancer, IL-15 / 1L- 15 sushi is separated from the first CAR and second CAR by a second cleavage site that flanks either of the two distinct CAR units. Methods of characterization of CAR can be seen in the references (Li et al, Nat Commun. 2023 Sep 22;14(l):5920; Chen et al. Leukemia. 2018 Feb; 32(2): 402-412). Each CAR unit has a VHH nanoantibody binding domain against BCMA or CD 19 antigen.

[0483] BCMA-CD19 ncCAR T demonstrated potent and selective cytotoxicity against BCMA or CD 19- positive cells in vitro. Co-culture assays revealed a remarkable lysis activity against BCMA or CD 19 positive cells, indicating effective dual-antigen targeting. In mouse models, both nCAR and ncCAR showed substantial cytotoxic activity, with depletion of BCMA and CD 19 positive cells and significant durable persistency. This ncCAR T represents a promising therapeutic strategy for patients with autoimmune diseases or malignancies who relapse after CAR therapy. The novel construct may provide improved efficacy and potential to re-dose a CAR relapsed patient.

[0484] BC4-AB19-N2-15R ncCAR T-cell Transduction Efficiency-To evaluate BC4-AB19-N2-15R ncCAR expression levels on the T-cell surface after retroviral transduction, flow cytometry analysis was used ( Figure 22 B). The transduction efficiency was determined by staining cells with goat- VHH antibody and analyzing them by flow cytometry. The transduction efficiency showed significant percentages of T cells expressing the BC4-AB19-N2-15R ncCAR construct (69.24%). BC4-AB19-N2-15R ncCAR T-cells effectively lyse plasma cell line expressing BCMA and B cell line expressing CD19- To evaluate the functional activity of BC4-AB19-N2-15R ncCAR T- cells, co-cultures were performed using the MM. Is (BCMA+) and REH (CD19+) cell lines.

[0485] Cells were stained with antibodies specific for BCMA (MM. Is) and CD3, or CD 19 (REH) and CD3, to distinguish between the target cells and effector T-cells (Figure 22C and 22D). Co- culture assays were performed at effector to target (E:T) ratios of 0.1 : 1 , 0.5 : 1 , and 1 : 1 for 8 hours, and flow cytometry analysis was used to determine cell lysis rates. BC4-AB19-N2-15R ncCAR T-cells demonstrated capability of elimination of both MM. Is and REH target cells at a very low E:T ratio.

[0486] BC4-AB19-N2-15R ncCAR T-cells exhibit significant anti-tumor activity in xenograft mouse models-In order to evaluate the in vivo functional activity of BC4-AB19-N2-15R ncCAR T- cells, a xenograft mouse model was developed using NSG mice. Mice were sublethally irradiated and injected with luciferase-expressing REH or U937-BCMA cells (Figure 22E and 22F). Six days following tumor cell injection, mice were treated with a course of 10 x 10e6 BC4-AB19- N2-15R ncCAR T-cells or vector control T cells. Tumor burden was monitored using bioluminescence imaging on days 5, 8, 11, 14, and 18. The bio luminescence images showed about 100% tumor reduction of REH or U937-BCMA tumor growth in mice treated with BC4- AB19-N2-15R ncCAR T-cells compared to control mice (Figure 22E and 22F).

[0487] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a BCMA antigen recognition domain (BCMA VHH) and second chimeric antigen receptor polypeptide having a CD19 recognition domain (CD19 VHH) linked to IL-15 / IL15sushi via a self-cleavage site. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 70, 84 and corresponding polynucleotide of SEQ ID NO. 69, 83

[0488] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a bi-specific BCMA and CD 19 antigen recognition domains (VHH-VHH) linked by a linker and targeting different antigens. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 74 and corresponding polynucleotide of SEQ ID NO. 73. In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a bi-specific BCMA and CD 19 antigen recognition domains (VHH-VHH) linked by a linker and co-expressing secreted IL-15 / IL15sushi. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 72 and corresponding polynucleotide of SEQ ID NO. 71.

[0489] Example for 19N10-19N2-BB ntCAR (bi-epitope CAR T cells)

[0490] In one embodiment, a bi-epitope CAR has a dual domain to bind two distinct epitopes on a antigen. This allows CAR T cells to target the interest cell with greater specificity and potentially overcome resistance by engaging multiple binding sites on the different part of CD 19 surface antigen.

[0491] In one embodiment, bi-epitope tandem CAR has a dual domain to bind two different antigens within one Bi-CAR. The structure of a dual domain is formed with VHH1-VHH2. Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain. The structure of the bispecific tandem CAR, 19N10-19N2-BB ntCAR (Figure 23A).

[0492] Based on the above-mentioned nanobody screening and targeting binding studies, anti-CD19 (N10) and anti-CD19 (N2) nanobodies antibodies were chosen as good candidates for generation of tandem CARs (ntCAR) targeting two different epitopes on the same antigen, CD19.

[0493] Peripheral blood mononuclear buffy coat cells were activated for three days and transduced with the anti-CD19 retroviral vectors for single CARs (19N10-BB nCAR. 19N10-BB nCAR) and ntCARs (19N10-19N2-BB ntCAR, N2N19 tan) or vector control. Expression of CAR on the T- cell surface was demonstrated three days after transduction by staining transduced T cells with goat anti-VHH antibody and mouse anti-human CD3. Three CARs, I9N2-BB nCAR (N2), 19N10-BB nCAR (N10), and 19N10-19N2-BB ntCAR (N2N10) showing similar CAR expression ( about 70%) in activated T cells were used to compare their killing activities of the target cells.

[0494] The assays involved co-culturing either control T cells or 19N2-BB nCAR (N2) T cells, 19N10-

[0495] BB nCAR (N10) CAR T cells and 19N10-19N2-BB ntCAR (N2N 10 Tan) T cells with REH cells expressing CD 19 at effector to target ( E:T) ratios of 0.1 : 1 , 0.5 : 1 , and 1 : 1 for 8 hours. Post- incubation, cells were stained with mouse anti-human CD3 and CD 19 antibodies and analyzed by flow cytometry. All three CAR T cells exhibited robust killing activities of elimination of target cells at effector to target (E:T) ratios of 0.5 : 1 , and 1 :1. 19N 10- 19N2-BB ntC AR T cells exhibited a superior anti-target cell effect with a lower E:T ratio of 0.1:1 than single-target CAR- T cells, I9N2-BB nCAR (N2) T cells or I9N10-BB nCAR (N 10) CAR T cells (Figure 23B).

[0496] Example for 19N10-19N2-28-15R ntCAR

[0497] In one embodiment, a bi-epitope CAR has a dual domain to bind two distinct epitopes on a antigen. This allows CAR T cells to target the interest cell with greater specificity' and potentially overcome resistance by engaging multiple binding sites on the different part of CD 19 surface antigen.

[0498] In one embodiment, bi-epitope tandem CAR has a dual domain to bind two different antigens within one Bi-CAR. The structure of a dual domain is formed with VHH1-VHH2. Bi-epitope tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain. The structure of the bi-epitope tandem CAR, 19N10-19N2-28- 15R ntCAR has anti-CD19 (N10) and anti-CD19 (N2) nanobodies targeting two epitopes on the same antigen, CD19(Figure 24A). A schematic showing the 19N10-19N2-28-15R ntCAR also equipped with a cytokine complex, IL-15 / IL-15sushi (also called 15R), and a self-cleaving sequence (P2A). Human peripheral blood T cells were activated and transduced with the retroviruses and the resulting in 19N10-19N2-28-15R ntCAR expression in the T cells. 19N10- 19N2-28-15R ntCAR T cells were then tested to analyze their functions using a co-culture assay with REH cells expressing CD19. Co-culture assay was performed at a effector to target (E:T) ratios of 0.1 :1, 0.5: 1 , and 1 : 1 for about 8 hours. Post-incubation, cells were stained with mouse anti-human CD3 and CD19 antibodies and analyzed by flow cytometry. 19N10-19N2-28-15R ntCAR T cells exhibits robust killing activity in a dose dependent manner and near complete elimination of all target cells at a low ratio of 1:1 (Figure 24B)

[0499] In particular embodiments, the engineered cell includes bi-epitope CD19 CAR linked to IL15 / IL- 15sushi via the P2A cleavage sequence. A polypeptide providing this embodiment includes SEQ ID No. 80 and corresponding polynucleotide sequence SEQ ID No.

[0500] 79

[0501] In an embodiment, the engineered cell includes bi-epitope CD19 CAR. A polypeptide providing this embodiment includes SEQ ID No. 82 and corresponding polynucleotide sequence SEQ ID No. 81

[0502] Example for BC4-ABI9-N2-BB-ntCAR

[0503] BC4-AB19-N2-BB-ntCAR has anti-BCMA (BC4) and anti-CD19 (AB 19-N2, also called N2) targeting BCMA and CD 19 antigens, respectively. Bispecific tandem CAR shares the leader sequence, hinge region, transmembrane domain, co-stimulatory domain(s) and CD3 zeta chain (Figure 25A). To evaluate BC4-AB19-N2-BB ntCAR expression levels on the T-cell surface after retroviral transduction, flow cytometry analysis was used (Figure 25B). The transduction efficiency was determined to be 76.38%.

[0504] BC4-AB19-N2-BB ntCAR T-cells Effectively Lyse MM. IS cell line (myeloma cell line expressing BCMA) and REH (expressing CD 19) Cell Lines-The killing activity of BC4-AB19- N2-BB ntCAR T-cells was assessed using co-culture assays with MM. IS and REH (CD 19+) cell lines (Figure 25C and 25D). Co-culture assays were performed at effector to target (E:T) ratios of 0.1: 1, 0.5:1, and 1 :1 for 8 hours. Flow cytometry analysis was used to determine cell lysis rates by BC4-AB19-N2-BB ntCAR T-cells or mock T-cells. BC4-AB19-N2-BB ntCAR T-cells demonstrated robust lysis of MM. IS and REH cells, achieving significant cytotoxicity across all tested ratios.

[0505] Example for 19N10-28 VAC nCAR

[0506] 19N 10-28 VAC nCAR consistent of a nanobody antibody VHH CD 19 nanobody antibody (also called 19N10), CD8 leader sequence. CD8 hinge and transmembrane regions, and a CD28 co- activator (also called CD28) fused to the CD3zeta signaling domain (Figure 26 A). The 19N 10-28 VAC nCAR also equipped with a cytokine complex, IL-15 / IL-15sushi (also called VAC), and a self-cleaving sequence (P2A). Activated T cells were transduced with retroviral 19N10-28 VAC nCAR. The transduced 19N10-28 VAC nCAR T cells effectively lyse target REH cells expressing CD19 in vitro - The cytotoxic potential of AB19-N2-28 VAC nCAR T cells was assessed by co-culturing them with REH tumor cells expressing CD 19 antigen. The assays involved co-culturing either control T cells or 19N10-28 VAC nCAR T cells with REH cells at effector to target ( E:T) ratios of 0.1 : 1 , 0.5:1, and 1 :1 for 8 hours. Post-incubation, cells were stained with mouse anti-human CD3 and CD19 antibodies and analyzed by flow cytometry. The results demonstrated that 19N10-28 VAC nCAR nCAR T cells completely lysed the target cells at all tested ratios, confirming their remarkable potent cytotoxic capability (Figure 26B).

[0507] Example for 19N10-BB nCAR

[0508] 19N10-BB nCA R consistent of a nanobody antibody VHH CD 19 nanobody antibody (also called 19N10 or N10), CD8 leader sequence, CD8 hinge and transmembrane regions, and a 4- IBB (also called BB) co-activator fused to the CD3zeta signaling domain (Figure 27A).

[0509] Figure 27B shows the transduction efficiency between activated T cells transduced with either a control retrovirus or the!9N 10-BB nCAR retroviruses, determined by labeling with goat-VHH antibody and streptavidin-PE conjugate. The analysis revealed that a significant percentage of T cells expressed 19N10-BB nCAR construct (about 75%, Figure 27B).

[0510] The 19N10-BB nCAR T cells were tested for their ability to lyse individual target cell lines in in vitro co-culture assays (Figure 27C). After 8-hour co-incubation, cells were labeled with anti- human CD3 and anti-human CD 19 and analyzed by flow cytometry. 19N10-BB nCAR T cells demonstrated the depletion of targeted of REH cells at effector to target (E:T) ratios of 0.5:1, and 1:1 for 8 hours of co-culture.

[0511] In order to evaluate the in vivo functional activity of 19N10-BB nCAR T cells, a xenograft mouse model was developed using NSG mice. Mice were sub lethally irradiated and injected with luciferase-expressing REH cells (Figure 27D). Six days following tumor cell injection, mice were treated with a course of 10 x 10Λ6 19N10-BB nCAR T cells B or vector control T cells. Tumor burden was monitored using bioluminescence imaging on days 4, 7, 1 1, 14, 18 and 33. The bioluminescence images showed about 100% tumor reduction of REH (expressing CD19) tumor growth in mice treated with 19N10-BB nCAR T cells compared to control mice (Figure 27D).

[0512] Example for Claudin nCAR (also called GI CAR)

[0513] Generation and Characterization of GI CAR-NK Cells- The GI CAR construct comprises an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a 4- IBB (BB) co-stimulatory domain, and a CD3g signaling domain. These components were designed to enable antigen-specific recognition, signal transduction, and activation of NK cell-mediated cytotoxicity (Figure 28 A).

[0514] CAR Expression in Transduced NK Cells-To assess the transduction efficiency and functionality of GI CAR-NK cells, purified NK cells from the cord blood buffy coat were transduced with a retroviral vector encoding the GI CAR construct or mock retroviral supernatant and incubated for five days. Following incubation, cells were harvested and stained with a goat anti- VHH antibody or goat IgG (control) at a dilution of 1 :250 for 30 minutes. Subsequently, cells were stained with anti-CD56 antibody and streptavidin-PE conjugate at 1 :500, fixed in 2% formalin, and analyzed by flow cytometry (Figure 28B). The results showed that 90.98% of GI CAR-NK cells expressed CAR, demonstrating efficient transduction and stable surface expression of the construct in NK cells.

[0515] Cytotoxicity of GI CAR-NK Cells Against SNU-601 Tumor Cells-To evaluate the tumor-killing activity of GI CAR-NK cells, a co-culture assay was performed using SNU-601 target cells, a gastric cancer cell line expressing Claudin 18.2. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells at different effector-to-target (E:T) ratios for 18 hours. After incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells (lower right columns) from target tumor cells (upper left colums), and analyzed by flow cytometry (Figure 28C). The flow cytometry analysis demonstrated that GI CAR-NK cells effectively lysed tumor cells in a dose-dependent manner. At an E:T ratio of 0.5: 1, GI CAR-NK cells killed 33% of the target cells, whereas at an E:T ratio of 1 : 1, the killing efficiency increased to 88%, confirming the potent cytotoxic function of GI CAR-NK cells in recognizing and eliminating Claudin 18.2-positive tumor cells. These findings demonstrate the ability of GI CAR-NK cells to efficiently target and kill gastric cancer cells, supporting their potential therapeutic applications.

[0516] Example for Claudin 18.2-IL15 / IL15sushi nCAR (also called GIV CAR)

[0517] Generation and Characterization of GIV CAR-NK Cells (Claudin 18.2 nCAR expressing 1L- 15 / IL15sushi)-The GIV CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a 4-1 BB (BB) co-stimulatory domain, a CD3L signaling domain, and a T2A self-cleaving sequence enabling the co-expression of the secreted IL-15 / IL-15sushi cytokine complex(Figure 29A).

[0518] CAR Expression in Transduced NK Cells-To confirm CAR expression, NK cells were transduced with either GIV CAR or mock viral supernatant and cultured for five days. After incubation, cells were harvested and stained with a goat anti-VHH antibody or goat IgG (control) at 1 :250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1 :500. Cells were then fixed in 2% formalin and analyzed by flow cytometry' (Figure 29B). The results demonstrated that 43.79% of GIV CAR-NK cells expressed CAR, confirming successful transduction and stable expression of the construct.

[0519] Cytotoxicity of GIV CAR-NK Cells Against SNU-601 Tumor Cells-To evaluate the tumor- killing capacity of GIV CAR-NK cells, a co-culture assay was conducted using SNU-601 target cells, a Claudin 18.2-expressing gastric cancer cell line. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells at different effector- to-target (E:T) ratios for 18 hours. Following incubation, cells were harvested, stained with anti- CD56 antibody to differentiate effector NK cells from target tumor cells, and analyzed by flow cytometry (Figure 29C). The flow cytometry' results demonstrated that GIV CAR-NK cells effectively lysed tumor cells, confirming their ability to recognize and eliminate Claudin 18.2- positive targets. At an E:T ratio of 0.5:1, GIV CAR-NK cells lysed 14% of tumor cells, while at an E:T ratio of 1 :1, the killing efficiency increased to 62%. These findings confirm that GIV CAR-NK cells exhibit strong cytotoxic activity. Example for Claudin 18.2-1L-18 nCAR (also called GI i 8 CAR)

[0520] Generation and Characterization of GI18 CAR-NK Cells- The GI18 CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a CD28 (28) co-stimulatory domain, a CD3g signaling domain, and a P2A self-cleaving sequence linked to the IL-18m cytokine. IL-18, a pro- inflammatory cytokine, is expected to enhance NK cell activation and anti-tumor function by promoting immune modulation and improving NK cell persistence (Figure 30A).

[0521] CAR Expression in Transduced NK Cells-To confirm the transduction efficiency and expression of the Gil 8 CAR construct, NK cells were transduced with either Gil 8 CAR or mock viral supernatant and cultured for five days. Cells were then harvested and stained with a goat anti- VHH antibody or goat IgG (control) at a dilution of 1:250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1 :500. Cells were subsequently fixed in 2% formalin and analyzed by flow cytometry (Figure 30B). The results showed that 79.26% of GI18 CAR-NK cells expressed CAR, confirming successful transduction and stable expression of the construct.

[0522] Cytotoxicity of GI18 CAR-NK Cells Against SNU-601 Tumor Cells-To evaluate the tumor- killing potential of Gil 8 CAR-NK cells, a co-culture assay was performed using SNU-601 target cells, a Claudin 18.2-expressing gastric cancer cell line. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells at different effector- to-target (E:T) ratios for 18 hours. After incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells from tumor target cells, and analyzed by flow cytometry (Figure 30C). The results demonstrated that GI18 CAR-NK cells effectively lysed tumor cells, confirming their strong cytotoxic potential. At an E:T ratio of 0.5: 1, GI 18 CAR-NK cells lysed 44% of target cells, and at an E:T ratio of 1: 1, the killing efficiency increased to 90%. These findings demonstrate the ability of GI18 CAR-NK cells to efficiently target and kill gastric cancer cells, supporting their potential therapeutic application.

[0523] Example for Claudin 18.2-1L-15 / 1L15sushi-lL-18 nCAR (also called GIVI 8 CAR) Generation and Characterization of GIVI 8 CAR-NK Cells-The GIVI 8 CAR construct consists of an anti-Claudin 18.2 nanobody (VHH, heavy chain-only antibody) as the antigen-binding domain, a hinge domain, a transmembrane (TM) region, a CD28 (28) co-stimulatory domain, and a CD3g signaling domain. Additionally, it includes self-cleaving sequences (T2A and P2A) that enable the co-expression of IL-15 / IL-15sushi and 1L-I8m cytokine modules. The inclusion of IL- 15 is expected to enhance NK cell proliferation and persistence, while IL- 18 contributes to immune modulation and further activation of NK cells (Figure 31 A).

[0524] CAR Expression in Transduced NK Cells-To confirm transduction efficiency and CAR expression, NK cells were transduced with either GIVI 8 CAR or mock viral supernatant and cultured for five days. After incubation, cells were harvested and stained with a goat anti-VHH antibody or goat IgG (control) at a dilution of 1 :250 for 30 minutes, followed by staining with anti-CD56 antibody and streptavidin-PE conjugate at 1:500. Cells were then fixed in 2% formalin and analyzed by flow cytometry (Figure 3 IB). The results demonstrated that 79.26% of GIVI 8 CAR-NK cells expressed CAR, confirming stable transduction and successful expression of the construct, similar to the GI18 CAR-NK group.

[0525] Cytotoxicity of GIVI 8 CAR-NK Cells Against SNU-601 Tumor Cells-To assess the tumor- killing potential of GIVI 8 CAR-NK cells, a co-culture assay was performed using SNU-601 target cells, a Claudin 18.2-expressing gastric cancer cell line. SNU-601 cells were pre-stained with CMTMR orange dye for 1 hour before being incubated with CAR-NK cells at different effector-to-target (E:T) ratios for 18 hours. After incubation, cells were harvested, stained with anti-CD56 antibody to distinguish effector NK cells from tumor target cells, and analyzed by flow cytometry (Figure 31 C ). The results demonstrated that GIVI 8 CAR-NK cells exhibited strong tumor-killing activity. At an E:T ratio of 0.5:1, GIV18 CAR-NK cells lysed 47% of the target cells, and at an E:T ratio of 1 :1, the killing efficiency increased to 90%. These findings indicate that the dual secretion of IL- 15 and IL- 18 enhances both NK cell survival and cytotoxic function, making GIV 18 CAR-NK cells the most effective construct tested. The synergistic effects of IL- 15 and IL- 18 contribute to increased persistence and immune activation, highlighting the therapeutic potential of GIVI 8 CAR-NK cells for Claudin 18.2-positive malignancies. Example for IL-15 and IL-18 secretion in CAR-NK Cells

[0526] To assess the cytokine secretion profiles of different CAR-NK constructs, ELISA was performed on culture supernatants collected after four days of incubation without exogenous cytokines.

[0527] IL-15 secretion by GIV CAR-NK cell-ELISA analysis revealed that GIV CAR-NK cells exhibited a higher level of IL- 15 as compared to that of IL- 15 secreted by GIVIS CAR-NK cells. In contrast, GI and GI18 CAR-NK cells showed negligible IL- 15 production, as expected, since these constructs lack the IL-15 module (Figure 32A). These results confirm that the IL-15 / IL- 15sushi module incorporated functionally into the GIV and GIV 18 CAR constructs.

[0528] IL- 18 Secretion by CAR-NK Cells-ln the evaluation of IL- 18 secretion, Gil 8 CAR-NK cells exhibited a higher IL- 18 level than that of GIVI 8 CAR-NK. Interestingly, GIVI 8 CAR-NK cells secreted 3.3-fold less IL-18 compared to Gil 8 CAR-NK cells (Figure 32B).

[0529] In Vitro Expansion of CAR-NK Cells-To evaluate the proliferative capacity of CAR-NK cells, transduced NK cells were cultured with IL- 15 and / or IL- 18 for seven days, followed by an additional culture period without cytokines until day 15. The expansion of CAR-NK cells was assessed using trypan blue staining at days 7, 11, and 15 to determine viable cei l counts (Figure 32C). The results demonstrated that GIV CAR-NK cells exhibited the highest expansion, reaching a 336-fold increase by day 15, followed by GI 18 CAR-NK cells, which showed a 264- fold expansion. Notably, GIVI 8 CAR-NK cells expanded 112-fold, indicating that while IL- 15 strongly promotes proliferation, the co-expression of IL- 15 and IL- 18 modulates expansion to maintain a controlled proliferation rate (Figure 32C). These findings further support the role of IL-15 and IL-18 as key regulators of CAR-NK expansion, with the dual-cytokine construct providing a more balanced cytokine profile to optimize NK cell proliferation and function.

[0530] In Vivo Anti-Tumor Efficacy of CAR-NK Cells

[0531] To investigate the therapeutic efficacy of CAR-NK cells, an NSG xenograft model was established using luciferase-expressing SNU-601 gastric cancer cells. Mice were sub lethally irradiated and intravenously injected with tumor cells to induce measurable tumor formation. Six days post- injection, mice were treated with 5 x 106CAR-NK cells or mock NK cells, and tumor burden was monitored using bioluminescence imaging on days 2, 13, 16, 30, and 42 (Figure 32D). Superior Tumor Regression in GIVI 8 CAR-NK-Treated Mice-Bioluminescence imaging revealed that CAR-NK-treated groups exhibited significant tumor regression compared to mock- treated mice, validating the effectiveness of CAR-NK therapy. Among the groups, GIVI 8 CAR- NK cells demonstrated the most substantial tumor reduction, with rapid tumor clearance observed as early as day 16 and near-complete tumor elimination by day 42 (Figure 32D). A quantitative analysis of tumor regression was performed to compare the efficacy of different CAR constructs (Figure 32D). Mice treated with GIVI 8 CAR-NK cells exhibited the most significant tumor reduction, confirming that the combination of IL- 15 and IL- 18 provides superior tumor control compared to single-cytokine constructs. The ability of GIV 18 CAR-NK cells to balance immune cell persistence while enhancing cytotoxicity highlights the synergistic effects of IL- 15 and IL- 18 secretion. These findings demonstrate that co-expression of IL- 15 and IL-18 enables enhanced tumor clearance while maintaining controlled immune cell expansion, addressing key challenges such as immune cell exhaustion and cytokine imbalance. This dual- cytokine CAR-NK platform offers a promising strategy for achieving safer and more effective CAR-based immunotherapy for Claudin 18.2-positive cancers.

[0532] In conclusion, unexpectedly, the co-expression of IL- 15 and IL- 18 in GIV 18 CAR-NK cells resulted in a synergistic effect, improving anti-tumor efficacy while maintaining a controlled proliferation rate. This controlled expansion mitigates the risks associated with excessive IL-15- driven proliferation, such as immune exhaustion, malignancy, and cytokine release syndrome (CRS). In vivo tumor regression studies further confirmed that GIV 18 CAR-NK cells exhibited superior tumor control compared to single-cytokine CAR constructs, demonstrating rapid tumor clearance and sustained therapeutic effects. The dual-cytokine CAR-NK approach in this invention provides a significant improvement over existing CAR-based therapies by enhancing immune cell persistence, optimizing cytokine balance, and ensuring sustained cytotoxic activity without inducing excessive proliferation. By addressing key challenges such as uncontrolled cytokine release, immune cell exhaustion, and limited persistence, this platform offers a safer, more effective, and broadly applicable CAR-NK therapy for treating Claudin 18.2-positive cancers and other malignancies. Accordingly, this invention discloses a novel CAR- cell therapy with a unique and optimized cytokine secretion profile, offering a superior strategy for maximizing therapeutic efficacy while minimizing risks. The combination of IL-15 and IL- 18 represents an innovative approach to enhancing immune cell functionality’ and improving CAR- based cancer immunotherapies, with potential applications across multiple tumor types and clinical settings.

[0533] In an embodiment, the engineered cell includes a Claudin 18.2 nCAR. A polypeptide providing this embodiment includes SEQ ID NO. 90 and corresponding polynucleotide sequence SEQ ID NO. 89

[0534] In particular embodiments, the engineered cell includes a Claudin 18.2CAR linked to IL- 18 via the P2A cleavage sequence. A polypeptide providing this embodiment includes SEQ ID NO. 94 and corresponding polynucleotide sequence SEQ ID NO. 93.

[0535] In some embodiments, the engineered cell includes a Claudin 18.2CAR linked to IL- 15 / IL15 susshi via the P2A cleavage sequence. A polypeptide providing this embodiment includes SEQ ID NO. 92 and corresponding polynucleotide sequence SEQ ID NO. 91.

[0536] In particular embodiments, the engineered cell includes a Claudin 18.2CAR linked to IL- 15 / IL15sushi and IL- 18 via the P2A self-cleavage sites. A polypeptide providing this embodiment includes SEQ ID NO. 96 and corresponding polynucleotide sequence SEQ ID NO. 95.

[0537] Example for N399-20h-19N2NI0-VAC ncCAR (also called N399) as a super CAR

[0538] The N399-20h-19N2N10-VAC ncCAR (N399) construct consists of two complete units of a humanized anti-CD20 single-chain variable fragment (scFv) CAR and a bi-epitope tandem CD 19 nCAR (VHH1-VHH2) binds two distinct epitopes on CD 19 antigen. Additionally, an IL-15 / IL- 15sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 2A). Each unit bear leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and co-stimulatory domain fused to a CD3c intracellular signaling domain. The CD20 CAR unit has CD28 co-stimulatory domain while the bi-epitope tandem CD19 nCAR bears 4-1BB co-stimulatory domain. Expression of the N399 CAR construct is controlled by a promoter, with two modular CAR units linked by a P2A self-cleaving peptide to ensure efficient expression. Additionally, an IL-15 / IL-15sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 33A).

[0539] In one embodiment, N399 has two complete units of a CD20 CAR and a CD 19 VHCAR that allows CAR T cells to target the interest cell with greater specificity and potentially overcome resistance by engaging multiple binding sites on the different surface antigens.

[0540] Transduction of T Cells with N399-2011-19N2N10-VAC CAR-Human T cells were activated and transduced with N399-2011-19N2N10-VAC CAR-encoding retroviral particles. Following five days of culture, flow cytometry analysis was performed to determine the expression efficiency. The results showed that 34.51% of transduced T cells expressed the CD20h CAR construct, while 69.89% expressed the CD19N2N10 CAR construct (Figures 33B and 33C).

[0541] ELISA assay for detecting IL- 15 Secretion in the N399 CAR T cells-To evaluate cytokine production, N399-20h-19N2N10-VAC CAR T cells were cultured at a density of 0.5 x 10Λ6 cells per well in AMI V medium for 72 hours. Culture supernatants were collected, and IL- 15 secretion was analyzed using an ELISA kit. The results showed elevated IL- 15 levels in the supernatants of N399 CAR T cells, while no detectable IL- 15 was observed in mock-transduced T cells (Figure 33D).

[0542] Cytotoxicity assay of N399-20h-19N2N10-VAC CAR T Cells-To assess cytotoxic activity, N399 CAR T cells were co-cultured with K562-CD20xp (expressing CD20) and K562-CD19xp (expressing CD19) target cells at an E:T ratio of 1 : 1 and 2:1. Target cells were pre-stained with CMTMR orange dye before being incubated with CAR T cells for 18 hours. Following co- culture, cells were stained with anti-CD3 antibody to differentiate effector cells from target cells and analyzed by flow cytometry (Figures 33E and 33F). The cytotoxicity assay demonstrated that N399-20h-19N2N10-VAC CAR T cells efficiently eliminated CD20- and CD19-expressing target cells. When co-cultured with K562-CD20xp cells at an effector-to-target (E:T) ratio of 1:1 and 2:1, N399-20h-19N2N10-VAC CAR T cells exhibited 34% and 82% target cell killing, respectively. Similarly, when N399-20h-19N2N10-VAC CAR T cells were co-cultured with K562-CD19xp cells, a target cell killing of 45% and 94% was observed at a 1 :1 and 2:1 E:T ratio, respectively. N409-20h-19N10-VAC CAR Construct (also called N409)

[0543] Schematic representation of N409-20h-19N10-VAC CAR construct is seen in Figure 34A.

[0544] The N40920h-19N10-VAC ncCAR (N409) construct consists of two complete units of a humanized anti-CD20 single-chain variable fragment (scFv) CAR and a CD19 VHH nCAR binds CD 19 antigen. Additionally, an 1L-15 / 1L-I5sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 34A). Each unit bear leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and co- stimulatory domain fused to a CD3g intracellular signaling domain. The CD20 CAR unit has CD28 co-stimulatory domain while the CD 19 VHH nCAR bears 4- IBB co-stimulatory domain. Expression of the N399 CAR construct is controlled by a promoter, with two modular CAR units linked by a P2A self-cleaving peptide to ensure efficient expression. Additionally, an IL-15 / IL- 15sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 34A).

[0545] The N409-20h-19N10-VAC chimeric antigen receptor (CAR) construct is designed to target both CD20- and CD19-expressing tumor cells by incorporating a humanized anti-CD20 single- chain variable fragment (scFv) and a CD19N10 nanobody (VHH) against CD 19. The expression of this dual CAR system is driven by a promoter, with the two modular CAR units linked by a P2A self-cleaving peptide, allowing for efficient co-expression of both targeting moieties. The structural organization of this CAR construct is illustrated in Figure 34A.

[0546] To assess the transduction efficiency of the N409-20h-19N10-VAC CAR, activated human peripheral blood-derived T cells were transduced with retroviral vectors encoding the CAR construct and subsequently expanded for five days. Flow cytometry analysis confirmed the successful expression of both CAR components within the transduced cells. The results demonstrated that 29% of the transduced T cells expressed the CD20h CAR construct, while 65.71% of the cells expressed the CD19N10 CAR construct. In contrast, mock-transduced control T cells exhibited negligible CAR expression, confirming the specificity and efficiency of transduction (Figure 34B-C).

[0547] The secretion of IL- 15 by N409-20h-19N10-VAC CAR T cells was evaluated to determine whether the IL- 15 / IL- 15 sushi domain incorporated within the construct facilitated T cell persistence. To quantify IL- 15 secretion, N409-20h-19N10-VAC CAR T cells were cultured at a density of 0.5 x 106cells per well for 72 hours, and the concentration of IL-15 in the culture supernatants was measured using an ELISA assay. The results demonstrated that the CAR- expressing T cells secreted a significant amount of IL- 15, whereas mock-transduced T cells produced undetectable levels of IL-15. These findings indicate that the inclusion of IL- 15 within the construct supports the survival and persistence of CAR T cells, potentially enhancing their therapeutic efficacy in vivo (Figure 34D).

[0548] The cytotoxic potential of N409-2011-19N10-VAC CAR T cells was assessed using K562- CD20xp and K562-CD19xp target cells, which were co-cultured with CAR T cells at different effector-to-target (E:T) ratios of 1:1 and 2:1. The target cells were pre-labeled with CMTMR orange dye, and after an 18-hour incubation period, their survival was analyzed by flow cytometry. The results indicated that CAR T cells efficiently eliminated CD20+ and CD 19+ target cells in a dose-dependent manner. Specifically, at an E;T ratio of 1:1, CAR T cells killed 59% of K562-CD20xp target cells and 66% of K562-CD19xp target cells. When the E:T ratio was increased to 2: 1, cytotoxicity was further enhanced, with 88% of K562-CD20xp and 99% of K562-CD19xp target cells eliminated. These results confirm that N409-20h-19N10-VAC CAR T cells exhibit strong antigen-specific cytotoxic activity against CD20- and CD19-expressing tumor cells, making them a promising therapeutic candidate for hematologic malignancies (Figure 34E and F).

[0549] N4I2-20h-19N2N10-18m CAR Construct (also called N412)

[0550] The N412-20h-19N2NI0-18m CAR construct is designed for dual targeting of CD20- and CD19-expressing tumor cells. The N412-20h-19N2N 10-VAC ncCAR (N412) construct consists of two complete units of a humanized anti-CD20 single-chain variable fragment (scFv) CAR and a bi-epitope tandem CD19 nCAR (VHH1-VHH2) binds two distinct epitopes on CD19 antigen. Additionally, an IL- 18 (also called IL-18M) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 35A). Each unit bears leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and co-stimulatory domain fused to a CD3g intracellular signaling domain. The CD20 CAR unit has a CD28 co-stimulatory domain while the bi-epitope tandem CD 19 VHH nCAR bears a 4- IBB co-stimulatory domain. Expression of the N399 CAR construct is controlled by a promoter, with two modular CAR units linked by a P2A self-cleaving peptide to ensure efficient expression. Additionally, an IL-15 / IL-15sushi domain (VAC) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 35A).

[0551] In one embodiment, N412 has two complete units of a CD20 CAR and a CD 19 VHCAR that allows CAR T cells to target the interest cell with greater specificity and potentially overcome resistance by engaging multiple binding sites on the different surface antigens.

[0552] To evaluate the transduction efficiency of the N412-20h-19N10N2-18m CAR, activated human peripheral blood-derived T cells were transduced with a retroviral vector encoding the CAR construct and subsequently expanded for five days. Flow cytometry analysis confirmed robust expression of both CAR components. The results demonstrated that 27.69% of the transduced T cells expressed the CD20h CAR construct, while 73.16% of the cells expressed the CD19N2N10 CAR construct. In contrast, mock-transduced control T cells exhibited negligible CAR expression, confirming the specificity and efficiency of transduction (Figures 35B and 35C)

[0553] To evaluate IL-18 secretion, N412-20h-19N2N 10-18m CAR T cells were seeded at a density of 0.5x106cells per well in a 96-well plate and incubated for 72 hours. Culture supernatants were collected, and IL- 18 levels were measured using an ELISA kit. The results confirmed robust IL- 18 secretion from N412 CAR T cells, whereas no detectable IL- 18 was observed in mock- transduced T cells, indicating successful cytokine production (Figure 35D).

[0554] To determine the cytotoxic activity of N412 CAR T cells, K562-CD20xp and K562-CD19xp target cells were used. Target cells were pre-stained with CMTMR orange dye and co-cultured with N412-20h-19N2N10-18m CAR T cells at an effector-to-target (E:T) ratio of 1: 1 and 2: 1. After an 18-hour incubation, cells were harvested, stained with anti-CD3 antibody, and analyzed via flow cytometry. The cytotoxicity assay demonstrated that N412-20h-19N2N10-18m CAR T cells efficiently eliminated CD20- and CD19-expressing target cells. When co-cultured with K562-CD20xp cells at an effector-to-target (E:T) ratio of 1 : 1 and 2:1, N412-20h-19N2N10-18m CAR T cells exhibited 21% and 67% target cell killing, respectively. Similarly, when N412-2011- 19N2N10-18m CAR T cells were co-cultured with K562-CD19xp cells, a target cell killing of 15% and 88% was observed at a 1:1 and 2: 1 E:T ratio, respectively (Figures 35E and 35F).

[0555] N413-20h- 19N10N2 CAR Construct (Also N413)

[0556] The N413-20h- 19N2N10-VAC ncCAR (N412) construct consists of two complete units of a humanized anti-CD20 single-chain variable fragment (scFv) CAR and a bi-epitope tandem CD 19 nCAR (VHH1-VHH2) binds two distinct epitopes on CD 19 antigen. Additionally, an IL- 18 (also called IL-18M) is incorporated downstream, connected via a T2A peptide sequence, allowing independent expression of both CARs and cytokines, thereby promoting T cell survival and persistence (Figure 35 A). Each unit bears leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and co-stimulatory domain fused to a CD3C intracellular signaling domain. The CD20 CAR unit has a CD28 co-stimulatory domain while the bi-epitope tandem CD19 VHH nCAR bears a 4-1BB co-stimulatory domain. Expression of the N399 CAR construct is controlled by a promoter, with two modular CAR units linked by a P2A self-cleaving peptide to ensure efficient expression (Figure 36A).

[0557] Human T cells were transduced with retroviral particles encoding the N413-20h-19N10N2 CAR construct. Cells were cultured for five days post-transduction before assessing CAR expression by flow cytometry. The results demonstrated efficient transduction, with 10.02% of cells expressing CD20h CAR and 49.13% expressing CD19N10N2 CAR, confirming successful retro viral-mediated gene delivery and expression (Figures 36B and 36C).

[0558] Cytotoxicity Assay of N413-20h-19N10N2 CAR T Cells

[0559] The cytotoxic activity of N413 CAR T cells was evaluated against K562-CD20xp and K562- CD19xp target cells. Target cells were labeled with CMTMR orange dye and co-cultured with CAR T cells at an effector-to-target (E:T) ratio of 2: 1 for 18 hours. After incubation, cells were stained with anti-CD3 antibody and analyzed via flow cytometry. The results showed that N413 CAR T cells effectively lysed CD20- and CD19-expressing target cells, with 31% killing of K562-CD20xp cells and 88% killing of K562-CD19xp cells (Figure 36D and E). These findings indicate that N413-20h-19N10N2 CAR T cells exhibit potent antigen-specific cytotoxic activity against tumor cells expressing CD20 and CD19

[0560] In an embodiment, N399 CAR or N409 CAR or N412 CAR or N413 CAR can be used to B cell lymphoma / leukemia or autoimmune diseases similar to those of hCD20-CD19 ncCAR described in the section of treating disease.

[0561] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a humanized CD20 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD 19 recognition domain (VHH). In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 104 and corresponding polynucleotide of SEQ ID NO. 103.

[0562] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a humanized CD20 antigen recognition domain and second chimeric antigen receptor polypeptide having a bi-epitope CD 19 recognition domains (VHH1- VHH2) linked to secreted IL-15 / IL15sushi via a self-cleavage site. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 98 and corresponding polynucleotide of SEQ ID NO. 97. In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a humanized CD20 antigen recognition domain and second chimeric antigen receptor polypeptide having a bi-epitope CD 19 recognition domains (VHH1- VHH2) linked to secreted IL- 18 via a self-cleavage site. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 102 and corresponding polynucleotide of SEQ ID NO. 101.

[0563] In one embodiment, the engineered cell includes a first chimeric antigen receptor polypeptide having a humanized CD20 antigen recognition domain and second chimeric antigen receptor polypeptide having camelid derived CD 19 recognition domains (VHH). In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 100 and corresponding polynucleotide of SEQ ID NO. 99.

[0564] Example for the screening and isolation of CLL1 (also called CLL-1, human C-type lectin-like molecule- 1)

[0565] Alpacas were immunized with human CLL1 protein. Peripheral blood mononuclear cell (PBMC) mRNA was isolated and processed into a VHH gene library, transformed into phage-competent bacteria to generate a bacteriophage library'. This library was panned against CLL1 antigens to enrich for binding clones, identified by high-throughput ELISA and cellular binding by flow cytometry using the same strategy described in Figure 1A. VHH candidates with binding greater than two-fold above background were sequenced. Using Biolayer Interferometry (BLI), association and dissociation rates were determined. Selected nanobodies were used to create a novel construct, termed CLLl nanobody CAR (CLLl nCAR).

[0566] Characterization of CLL 1 -Specific Nanobody (VHH) Candidates- Three leading anti-CLLl VHH candidates, LL50, LL56, and LL88, were selected for detailed physicochemical analysis (Figure 1). The amino acid sequences of VHH antibodies against CLLl are shown in Table 21, 22 and 23. These nanobody variants exhibited distinct molecular weights ( 12.17-13.45 kDa) and amino acid lengths (113-123 residues). The isoelectric points (pl range 5.24-7.97) varied across candidates, influencing solubility and pH-dependent stability. Extinction coefficient analysis under reduced and cystine-bridged conditions was performed to evaluate nanobody folding properties and disulfide bond integrity. The extinction coefficients ranged from 1.5163 to 2.3992, indicating differences in structural stability and folding under physiological conditions.

[0567] Expression and Purification of CLL1 -Specific Nanobody antibodies (VHH) Candidates-examples of purified CLL-1 -specific nanobody antibodies, such as LL50, LL56, and LL88 were subjected to SDS-PAGE analysis under reducing conditions to assess purity and molecular weight (Figure 37A). The gel analysis revealed distinct protein bands at the expected molecular weights (-12-14 kDa) for each VHH candidate. Coomassie Brilliant Blue staining confirmed the absence of major contaminating proteins, demonstrating successful expression and purification of high-purity CLL1 -specific VHHs. To further evaluate the structural homogeneity of the purified nanobodies, size-exclusion chromatography (SEC) was performed for each candidate (Figures 37A). Chromatographic analysis showed sharp, single peaks for LL50, LL56, and LL88, indicating that each nanobody exists predominantly as a monomer in solution.

[0568] Table 21: FR and CDR amino acid sequences of a LL50 VHH nanobody antibody for CLL1

[0569] Table 22: FR and CDR amino acid sequences of a LL56 VHH nanobody antibody for CLL1

[0570] Table 23: FR and CDR amino acid sequences of a LL88 nanobody antibody for CLL1 Cell-Based Binding Assay of CLL 1 -Specific Nanobody (VHH) Candidates-To assess the binding efficiency of CLL1 -specific nanobody (VHH) candidates, a cell-based binding assay was conducted using HEK293 cells overexpressing CLL1. The binding affinity of LL50, LL56, LL88, and additional VHH variants was analyzed using flow cytometry, where each nanobody was incubated with CLL 1 -expressing HEK293 cells, followed by staining with a fluorophore- conjugated secondary antibody to detect binding interactions. The binding responses were recorded across a range of nanobody concentrations to determine the half-maximal effective concentration (EC50) values, which serve as an indicator of binding affinity'. Flow cytometry analysis revealed dose-dependent binding of the nanobody candidates to CLL1 -overexpressing HEK293 cells, confirming specific interactions between each nanobody and its target (Figure 37B). The binding curves demonstrated variability in the interaction strength of different VHH candidates with the CLL1 target. To quantitatively compare the binding affinity among the candidates, EC50 values were determined for LL50, LL56, LL88, and additional VHH candidates. The results showed that LL50, LL56, and LL88 exhibited lower EC50 values, indicating a higher binding affinity for CLL1 -expressing cells compared to other tested nanobody candidates. Specifically, LL50 demonstrated an EC50 of 0.645 nM, LL56 exhibited the highest affinity with an EC50 of 0.1443 nM, and LL88 displayed an EC50 of 0.9389 nM. Among these candidates, LL56 showed the strongest binding affinity.

[0571] ELISA-Based Binding Assay of CLL1 -Specific Nanobody (VHH) Candidates-To determine the binding affinity of CLL 1 -specific nanobody (VHH) candidates, an enzyme-linked immunosorbent assay (ELISA) was conducted using recombinant human CLL 1 -His protein immobilized onto ELISA plates. (Figure 37C) The VHH candidates, including LL50, LL56, LL88, and additional variants, were incubated with the immobilized CLL1 protein, followed by detection using an HRP- conjugated secondary' antibody. The binding interactions were quantified through an optical density (OD) measurement, and the resulting binding curves were used to assess the dose- dependent interaction of each nanobody with human CLL 1.

[0572] The half-maximal effective concentration (EC50) values were calculated for LL50, LL56, LL88, and additional VHH candidates to provide a quantitative measure of binding affinity (Figure 4). The results demonstrated that LL50 exhibited an EC50 of 0.2883 nM, LL56 had an EC50 of 0.4047 nM, and LL88 displayed an EC50 of 0.494 nM. These values indicate that LL50 and LL56 had the higher binding affinity' for human CLLl-His protein, followed by LL88, while other VHH candidates showed varying degrees of binding efficiency.

[0573] Biolayer Interferometry (BLI) Binding Kinetics Analysis of CLL1 -Specific Nanobody (VHH) Candidates-To evaluate the binding kinetics of CLL1 -specific nanobody (VHH) candidates, biolayer interferometry (BLI) analysis was performed using human CLLl-His protein immobilized onto biosensor surfaces (Figure 5). The VHH candidates, including LL50, LL56, LL88, and additional variants, were tested at varying concentrations to assess their association and dissociation kinetics. The interaction between each nanobody and CLL1 was measured for the association rate constant (Ka), dissociation rate constant (Kdis), and equilibrium dissociation constant (KD).

[0574] Among the tested nanobody candidates, LL56 demonstrated the highest binding affinity, with a KD value of 1.16E-09 M. The data collectively demonstrate that LL56 binds to CLL1 with the highest affinity and the slowest dissociation rate, making it the most promising candidate for therapeutic applications. LL50 and LL88 also exhibited strong binding, though with higher KD values and faster dissociation rates, indicating moderate interaction compared to LL56. The results confinn that LL56, LL50, and LL88 exhibit strong and specific binding interactions with CLL1.

[0575] Example for N414-LL56-33B nCAR (Also called CLL1-CD33 ncCAR)

[0576] Schematic Representation of N414-LL56-33B CAR Construct (Figure 38A). The N414-LL56-33B CAR construct consists of a an anti-CLLl LL56 nanobody (VHH) against CLL1 and anti-CD33 single-chain variable fragment (scFv) against CD33, enabling dual targeting of CLL1- and CD33- expressing tumor cells. The construct includes two complete units of CARs. Each bear leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and a 4- IBB (in CLL CAR) or CD28 ( in CD33 CAR) co-stimulatory domain fused to a CD3L intracellular signaling domain to enhance T cell activation. Expression of the CAR construct is controlled by a promoter, with two modular CAR units linked by a P2A self-cleaving peptide, allowing independent expression of both CARs (Figure 38A). Functional Cytotoxicity Assay of N414-LL56-33B CAR T Cells- activated human peripheral blood-derived T cells were transduced with retroviruses encoding the CAR construct and subsequently expanded for five days. Flow cytometry analysis confirmed the successful expression of both CAR components within the transduced cells. The transduced T cells were then used for cytotoxicity assays. Cytotoxicity assays were performed using REH-CLLlxp (CLL1 -overexpressing) and REH-CD33xp (CD33 -overexpressing) target cells. Target cells were co-cultured with CAR T cells at effector-to-target (E:T) ratios of 2: 1 and 4: 1. After an 18-hour incubation, cells were harvested, washed, stained with anti-CLLl or anti-CD33 antibody in combination with anti-CD3 antibody to distinguish effector cells from target cells, and analyzed via flow cytometry (Figure 38B and C). The cytotoxicity assay demonstrated that N414-LL56- 33B CAR T cells efficiently eliminated CLL1- and CD33 -expressing target cells. When co- cultured with REH-CLLlxp cells at an effector-to-target (E:T) ratio of 2: 1 and 4: 1, N414-LL56- 33B CAR T cells exhibited 55% and 82% target cell killing, respectively. Similarly, when N414- LL56-33B CAR T cells were co-cultured with REH-CD33xp cells, a target cell killing of 75% and 95% was observed at a 2:1 and 4:1 E:T ratio, respectively.

[0577] Example for N415-LL56-33B-18m CAR T Cells (Also called CLL1-CD33-IL18 cnCAR) N415-LL56-33B-18m CAR Construct Design is described in Figure 39A. The N415-LL56-33B- 18m CAR construct was designed to enable dual targeting of CLL1- and CD33-expressing tumor cells, with additional IL- 18 (also called IL- 18m) incorporation to enhance T cell expansion, persistence, and tumor-killing activity. This construct consists of an anti-CLLl LL56 nanobody (VHH) against CLL1 and an anti-CD33 single-chain variable fragment (scFv) against CD33, linked by a P2A self-cleaving peptide, allowing for independent expression of both complete uits of CARs. Each unit of CAR design includes leader sequence, antigen recognition domain, hinge regions, transmembrane (TM) domains, and a 4-1BB or CD28 co-stimulatory domain fused to a CD3g intracellular signaling domain, enhancing T cell activation and persistence. Additionally, the secreted IL- 18m domain is expressed as a separate unit through a T2A self-cleaving peptide, facilitating cytokine-mediated augmentation of CAR T cell function. The expression is controlled by a single promoter (Figure 39A). Cytotoxicity of N415-LL56-33B-18m CAR T Cells Against Target Cells- activated human peripheral blood-derived T cells were transduced with retrovirues encoding the CAR construct and subsequently expanded for about five days. Flow cytometry analysis confirmed the successful expression of both CAR components within the transduced cells. The transduced T cells were then used for cytotoxicity assays. The functional cytotoxic activity of N415-LL56- 33B-18m CAR T cells was assessed against CLL1- and CD33 -expressing tumor cells using an in vitro cytotoxicity assay. REH-CLLlxp (CLLl-overexpressing) and REH-CD33xp (CD33- overexpressing) target cells were co-cultured with N415-LL56-33B-18m CAR T cells at effector-to-target (E:T) ratios of 2: 1 and 4: 1 for ...

Claims

Claims:

1. An isolated camelid nanobody antibody (VHH), which binds specifically to human CD 19. BCMA, Claudin 18.2 or CLL1 wherein the antibody that binds to CD 19 comprises SEQ ID NO: 18, SEQ ID NO: 20, 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, Er SEQ ID NO. 32; wherein the antibody that binds to BCMA comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:

8. , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; wherein the antibody that binds to Claudin 18.2 comprises SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40; wherein the antibody that binds to CLL1 comprises SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 128; and wherein the antibody binds to CDR1. CDR2, and CDR3 amino acid sequences that are at least 80% identical to CDR1 , CDR2, and CDR3 sequences in any one of CD 19, BCMA, Claudin 18.2 and CELL2. A recombinant polypeptide comprising one or more VHH antibodies according to claim 1.

3. A multivalent antibody comprising a first antigen-binding moiety comprising a VHH antibody according to claim 1 and a second antigen-binding portion.

4. The multivalent antibody of claim 3, wherein the second antigen-binding moiety binds specifically to a cell-surface marker of an immune cell.

5. The multivalent antibody of claim 3, wherein the second antigen-binding moiety binds specifically to a cell-surface marker of an immune cell including T and NK cells.

6. The multivalent antibody of claim 3, wherein the second antigen-binding moiety binds specifically to CD3.

7. The multivalent antibody of any one of claims 3 to 6, wherein said VHH antibody comprises any SEQ ID NO of claim 1.

8. An engineered chimeric antigen receptor polypeptide (CAR) that binds to CD19 (anti-CD19 CAR) or BCMA (anti-BCMA CAR) or Claudin 18.2 (anti-Claudin 18.2) or CLL1 (anti-CLLl), wherein said CAR comprises a signal peptide (leader sequence), a camelid-derived antigen recognition domain (VHH), a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.

9. The engineered chimeric antigen receptor polypeptide (CAR) according to claim 8, wherein the CDR1 to CDR3 of said anti-CD19 CAR camelid-derived antigen recognition domain (VHH) domain (VHH) is selected from SEQ ID NO:

18. SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ, SEQ ID NO: 30, or SEQ ID NO: 32, and wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 76 or SEQ ID NO: 78 and the corresponding nucleotide sequences of SEQ ID NO: 67, SEQ IDNO: 75 or SEQ ID NO: 77, respectively.

10. The engineered chimeric antigen receptor polypeptide (CAR) according to claim 8, wherein said anti-CD19 CAR camelid-derived antigen recognition domain (VHH) comprises a bi-epitope tandem two anti-CD19 antib Edies c nnected by a linker; wherein each anti-CD19 antibody binds to a different part of the CD 19 antigen; and wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 82 and the corresponding nucleotide sequences of SEQ ID NO: 81.

11. The engineered chimeric antigen receptor polypeptide (CAR) according to claim 8, wherein the CDR1 to CDR3 of camelid-derived antigen recognition domain (VHH) and camelid-derived antigen recognition domain (VHH) of said anti-BCMA CAR is selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or SEQ ID NO: 16; wherein the anti- BCMA CAR comprises the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 88 and the corresponding nucleotide sequences of SEQ ID NO: 85 or SEQ ID NO: 87, respectively.

12. The engineered chimeric antigen receptor polypeptide (CAR) according to claim 8, wherein the CDR1 to CDR3 of camelid-derived antigen recognition domain (VHH) and camelid-derived antigen recognition domain (VHH) of said anti-claudin 18.2 CAR is selected from SEQ ID NO: 34, , SEQ ID NO: 36, SEQ ID NO: 38, or SEQ ID NO: 40, and wherein the anti-claudin 18.2 CAR comprises the amino acid sequence of SEQ ID NO: 90 and the corresponding nucleotide sequences of SEQ ID NO: 89.

13. The engineered chimeric antigen receptor polypeptide (CAR) according to claim 8, wherein the CDR1 to CDR3 of camelid-derived antigen recognition domain (VHH)and camelid-derived antigen recognition domain (VHH) of said anti-CLLl CAR is selected from of SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ IDNO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, or SEQ ID NO: 128.

14. An engineered cell co-expressing two distinct chimeric antigen receptor (CAR) units at the cell surface, wherein the engineered cell comprises a nucleotide sequence comprising from 5' to 3' a first polynucleotide encoding a first chimeric antigen receptor (CAR) polypeptide, a second polynucleotide encoding a second chimeric antigen receptor (CAR) polypeptide, and a nucleotide encoding a viral self-cleavage peptide disposed between the first CAR polypeptide and the second CAR polypeptide, wherein a two unit CAR is formed, wherein the first and second CAR polypeptides are under the transcriptional control of a single promoter, wherein the first CAR polypeptide comprises a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co- stimulatory domain, and a first CD3 ζ signaling d main; and the second CAR polypeptide comprises a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co- stimulatory domain, and a second CD3ζ signaling d main; and wherein the first antigen recognition domain and the second antigen recognition domain are different and each bind to a different target, wherein the target of the first antigen recognition domain and second antigen recognition domain are, irrespective of order, CLL1 and CD33, wherein the first and second co-slimulaloiy domains are intracellular, and wherein the cleavagesite is selected from the group consisting of porcine teschovirus- 1 2A (P2A), thoseaasigna virus 2A (T2A), equine rhinitis A virus (ERAV) 2A (E2A), and FMDV 2A (F2A); and wherein the two-unit CAR comprises the amino acid sequence of SEQ ID NO: 122 and the corresponding nucleotide sequences of SEQ ID NO: 121.

15. An engineered cell co-expressing two distinct chimeric antigen receptor (CAR) units at the cell surface, wherein the engineered cell comprises a nucleotide sequence comprising from 5' to 3' a first polynucleotide encoding a first chimeric antigen receptor (CAR) polypeptide, a second polynucleotide encoding a second chimeric antigen receptor (CAR) polypeptide, and a nucleotide encoding a viral self-cleavage peptide disposed between the first CAR polypeptide and the second CAR polypeptide, wherein a two unit CAR is formed, wherein the first and second CAR polypeptides are under the transcriptional control of a single promoter, wherein the first CAR polypeptide comprises a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co- stimulatory domain, and a first CD3 signaling d main; and the second CAR polypeptide comprises a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co- stimulatory domain, and a second CD3C signaling d main; and wherein the first antigen recognition domain and the second antigen recognition domain are different and each bind to a different target, wherein the target of the first antigen recognition domain and second antigen recognition domain are, irrespective f rder, CD20 and CD19; wherein CD 19 antigen recognition comprises bi-epitope two tandem camelid derived nanobodyantibodies; wherein the first and second cN-stimulatory domains are intracellular, and wherein the cleavage site is selected from the group consisting of porcine teschovirus-1 2A (P2A), th seaasigna virus 2A (T2A), equine rhinitis A virus (ER AV) 2A (E2A), and FMDV 2A (F2A); and whereas the two-unit CAR comprises the amino acid sequence of SEQ ID NO: 104 and the corresponding nucleotide sequences of SEQ ID NO: 103.

16. The engineered cell according to any one of claims 8 to 15, further comprising at least one enhancer selected from the group consisting of PD- 1, PD-L1, CSFIR, CTAL-4, TIM-S, TGFR beta, IL- 2, IL-7, IL-12, IL-15, IL-15 / IL15sushi, IL-18 and IL-21 functional fragments thereof, or combinations thereof or a functional fragment thereof.

17. The engineered cell according to any one of claims 8 to 15, further comprising one or two enhancers of secreted IL-15 / IL15sushi and secreted IL- 18 functional fragments thereof, or combinations thereof or a functional fragment thereof.

18. An engineered cell co-expressing two distinct chimeric antigen receptor (CAR) units at the cell surface and secreted IL-15 / IL15sushi, wherein the engineered cell comprises a nucleotide sequence comprising from 5' to 3' a first polynucleotide encoding a first chimeric antigen receptor (CAR) polypeptide, a second polynucleotide encoding a second chimeric antigen receptor (CAR) polypeptide, and a nucleotide encoding a viral self-cleavage peptide disposed between the first CAR polypeptide and the second CAR polypeptide, forming a two unit CAR unit, wherein the first and second CAR polypeptides are under the transcriptional control of a single promoter, wherein:(i.) the first CAR polypeptide comprises a first antigen recognition domain (VHH), a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first CD3 ζ signaling d main; and(ii.) the second CAR polypeptide comprises a second antigen recognition domain (VHH), a second signal peptide, a second hinge region, a second transmembrane domain, a second co- stimulatory domain, and a second CD3g signaling dEmain; and wherein the first antigen recognition domain and the second antigen recognition domain are different and each bind to a different target, wherein the target of the first antigen recognition domain and second antigen recognition domain are, irrespective of order, BCMA and CD 19, wherein the first and second co-stimulatory domains are intracellular, and wherein the cleavage site is selected from the group consisting of porcine teschovirus-1 2A (P2A), thoseaasigna virus 2A (T2A), equine rhinitis A virus (ERAV) 2A (E2A), and FMDV 2A (F2A); and wherein said two unit CAR comprises the amino acid sequence of SEQ ID NO: 70 and SEQ ID NO: 84 and the corresponding nucleotide sequences of SEQ ID NO: 69 and SEQ ID NO: 83, respectively.

19. An engineered cell co-expressing a CAR comprising tandem camelid derived anti- BCMA and anti-CD19 nanobody antibodies (antigen recognition domains) connected by a linker, a hinge domain, transmembrane (TM) regions, and co- activation domains linked to the CD3C signaling dEmain; wherein said CAR comprises the amino acid sequence of SEQ ID NO: 74 and the corresponding nucleotide sequence of SEQ ID NO: 73.

20. The engineered cell of claim 9, further comprising secreted IL-15 / IL-15sushi and a camelid anti-CD19 VHH CAR, wherein the camelid anti-CD19 VHH CARcomprises the amino acid sequence of SEQ ID NO: 66 and the corresponding nucleotide sequence of SEQ ID NO: 65.

21. The engineered cell of claim 9, further comprising secreted IL-15 / IL-15sushi and a camelid anti-CD19 VHH CAR, wherein the camelid anti-CD19 VHH CAR comprises the amino acid sequence of SEQ ID NO: 66 and the corresponding nucleotide sequence of SEQ ID NO: 6522. The engineered CAR of claim 10, further comprising IL-15 / IL-15sushi and a camelid bi-epitope anti-CD19 VHH CAR, wherein the camelid bi-epitope anti- CD 19 VHH CAR comprises the amino acid sequence of SEQ ID NO: 80 and the corresponding nucleotide sequence of SEQ ID NO: 79.

23. The engineered CAR of claim 11, further comprising IL-15 / IL-15sushi and a camelid anti-BCMA VHH CAR, wherein the camelid anti-BCMA VHH CAR comprises the amino acid sequences of SEQ ID NO: 61 and SEQ ID NO: 64 and the corresponding nucleotide sequences of SEQ ID NO: 60 and SEQ ID NO: 63, respectively.

24. The engineered CAR of claim 12, further comprising IL- 15 / IL- 15sushi and a camelid anti-claudin 18.2 VHH CAR, wherein the camelid anti-claudin 18.2 VHH CAR comprises the amino acid sequence of SEQ ID NO: 92 and the corresponding nucleotide sequence of SEQ ID NO: 91.

25. The engineered cell of claim 12, further comprising IL- 18 and a camelid anti- claudin 18.2 VHH CAR, wherein the camelid anti-claudin 18.2 VHH CARcomprises the amino acid sequence of SEQ ID NO: 94 and the corresponding nucleotide sequences of SEQ ID NO: 93.

26. The engineered CAR of claim 12, further comprising secreted IL-18 and secreted IL-15 / IL15sushi and a camclid anti-c laudin 18.2 VHH CAR, wherein the camelid anti-claudin 18.2 VHH CAR comprises the amino acid sequence of SEQ ID NO: 96 and the corresponding nucleotide sequences of SEQ ID NO: 95.

27. The engineered cell of claim 14, further comprising secreted lL-15 / IL15sushi and a compound CAR targeting CLL1 and CD33, and comprising the amino acid sequence of SEQ ID NO: 126 and the corresponding nucleotide sequence of SEQ ID NO: 125.

28. The engineered cell of claim 14, further comprising secreted IL- 18 and a compound CAR targeting CLL1 and CD33,and comprising the amino acid sequence of SEQ ID NO: 124 and the corresponding nucleotide sequence of SEQ ID NO: 123.

29. The engineered cell of claim 15, further comprising secreted IL-15 / IL15sushi and a compound CAR targeting CD20 and CD 19, wherein said CD 19 antigen recognition domain comprises bi-epitop tandem CD 19 nano antibodies and comprises the amino acid sequence of SEQ ID NO:97 and the corresponding nucleotide sequence of SEQ ID NO: 98.

30. The engineered cell of claim 15, further comprising secreted IL-15 / IL15sushi and a compound CAR targeting CD20 and CD 19, wherein said CD 19 recognition domain comprises a bio-epitope comprising two camelid derived antibodies, and whereinsaid compound CAR comprises the amino acid sequences of SEQ ID NO:98 and the corresponding nucleotide sequences of SEQ ID NO: 97.

31. The engineered cell of claim 15, further comprising secreted IL-15 / IL15sushi and a c mp und CAR targeting CD20 and CD 19; wherein said CD 19 recognition domain comprises a camelid derived nanobody antibody, and wherein said compound CAR comprises the amino acid sequence of SEQ ID NO:99 and the corresponding nucleotide sequence of SEQ ID NO: 100.

32. The engineered cell of claim 15, further comprising secreted IL- 18 and a compound CAR targeting CD20 and CD 19; wherein said CD 19 recognition domains comprises a bio-epitope comprising two camelid derived antibodies, and wherein said compound CAR comprises the amino acid sequences of SEQ ID NO: 102 and the corresponding nucleotide sequences of SEQ ID NO: 101.

33. A method of treating a cell proliferative disease in a patient in need thereof comprising administering an engineered cell according to anyone of claims 8-10, 15, 18-23 or 29-32 to said patient, wherein the cell proliferative disease is selected from the group consisting of B-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia cells, primary effusion lymphoma, reticulohistiocytoma, lymphocyte predominant Hodgkin's lymphoma, and post- transplant lymphoproliferative disorders.

34. A method of eliminating or reducing B cells, immature B cells, memory B cells, plasmablasts, long lived plasma cells, or plasma cells associated with autoimmune disease in a patient in need thereof comprising administering an engineered cellaccording to any one of claims 8-10, 15, 18-23 or 29-32 to said patient, wherein the autoimmune disease is selected from the group consisting of, but not limited to achalasia, Addison’s disease, acute inflammatory demyelinating polyneuropathy - AIDP, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM7anti-TBM nephritis, anti-PAD4-activating rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticarial, axonal & neuronal neuropathy (AMAN), Balo disease, Behcet’s disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman disease (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan’s syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, CREST syndrome, Crohn’s disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic’s disease (neuromyelitis optica), diabetes mellitus, discoid lupus, Dressier’ s syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture’s syndrome, granulomatosis with polyangiitis, Graves’ disease, Guillain-Barre syndrome,Hashimoto’s disease, Hashimoto’s thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), hypogammalglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, lgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (TTP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritisjuvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, ligneous conjunctivitis, linear IgA disease (LAD), lupus, lyme disease chronic, membranous nephropathy, Meniere’s disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndromes types I, II, and III, polymyalgia rheumatic, polymyositis, postmyocardial infarction syndrome, postpericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis,psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud’s syndrome, reactive Arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, sensitized / preformed antibodies in solid organ transplant, Sjogren’s syndrome, sperm & testicular autoimmunity, stiff person syndrome (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac’s syndrome, sympathetic ophthalmia (SO), Takayasu’s arteritis, temporal arteritis / Giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi- Harada disease; and Wegener’s disease. Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N- methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorders (MOGSD), neuromyelitis optica spectrum (NMOSD), myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis.

35. A method of treating a cell proliferative disease in a patient in need thereof comprising administering an engineered cell according to any one of claims 11 and18-19 to said patient, wherein the cell proliferative disease is selected from thegroup consisting of plasma cell neoplasms is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom’s macroglobulinema, heavy chain diseases, solitary' bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.

36. A method of treating a cell proliferative disease in a patient in need thereof comprising administering an engineered cell according to any one of claims 12 and 24-26 to said patient, wherein the cell proliferative disease is selected from the group consisting of gastric cancers, breast cancers, colon cancers, liver cancers and pancreatic cancers.

37. A method of treating a cell proliferative disease in a patient in need thereof comprising administering an engineered cell according to any one of claims 13-14 and 27-28, to said patient, wherein the cell proliferative disease is selected from the group consisting of acute myeloid leukemia, chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, myelomonocytic leukemia, and myelodysplastic syndromes (MDS).

38. A method according to any one of claims 33-37 further comprising co- administration of chemotherapeutics agents, radiations, immunotherapy, surgery, and immunosuppressor agents.