Technical Field

The present disclosure relates to an engineered chimeric antigen receptor, which is capable of interacting with Cluster of Differentiation 19 molecule (CD 19) expressed on the surface of B- lymphocytes or cells of B-lymphocytes lineage. Further, the present disclosure also includes genetically engineered cells, particularly immunogenic T cells, being conferred with the ability to express and carry the chimeric antigen receptor.

Background

B-cell acute lymphoblastic leukemia (B-cell ALL) is the most common childhood cancer and it has an overall good prognosis, with up to 90% of the patients being alive at 5 years after diagnosis. ¹ However, for those who relapse on therapy, or who suffer chemotherapy -refractory disease, the outlook is poor. Allogeneic hemopoietic stem cell transplantation (allo-HSCT) still offers a chance for cure, but unfortunately relapse is common after allo-HSCT for leukemia. Patients who relapse ALL after allo-HSCT are very difficult to manage, and in addition to chemotherapy donor lymphocyte infusions have been advocated as an effective adjunct by many investigators, although available data do not support their use in patients with overtly active disease. Therefore, new treatment strategies for relapsed/refractory B- cell ALL are essential. In this context, CD 19 is a highly attractive target for immunotherapy as its expression is virtually universally present on the blasts in B-cell ALL, but it is otherwise restricted to normal cells of the B lineage. ' Modification of the T-cell receptors for the CD 19 anti- gen subsequently led to the development of chimeric antigen receptor (CAR) T-cell preparations for treatment of refractory B-cell ALL and advanced B-cell lymphomas. ³

The basic structure of the CAR-modified T cell is composed of an extracellular antigen-binding domain derived from a monoclonal antibody, and the signaling domains from the T-cell receptor to ren- der a capability of targeting a specific surface antigen(s). The second- and third-generation CARs incorporate one or two co-stimulatory molecules such as CD28, 4- 1BB, or OX40, ⁴ which promotes in vivo expansion. ⁵ In addition to the co-stimulatory molecules, another element of CARs, a spacer domain (CH2CH3), appears to play an important role in mediating antitumor activity. It has been reported that CARs with a mutated CH2 region or without CH2 region confer T-cell persistence and more potent antitumor responses in murine models. ⁴ Other investigators demonstrated the role of CAR spacer length in binding of the receptor to its target antigen. ^6.7 Adoptive T-cell trans- fer of such CAR-modified T cells has become a new frontier in cancer immunotherapy. The field rapidly advanced into multiple clinical trials and led to regulatory approval of the first two CAR T-cell preparations for treatment of B-cell ALL and refractory lymphomas by the US FDA. ^S Indeed, CD19 CAR T cells have demonstrated exceptional antitumor effects with up to 80% of the patients achieving complete remission (CR) in some trials. ^5,9,10 However, emerging safety issues regarding CAR T-cell therapy should be carefully considered. Toxicides following CAR T-cell infusions include cytokine release syndrome (CRS), neurologic toxicity, and “on target/off tumor” recognition, which are among the mos t serious and can turn into life-threatening or even lethal adverse events. ¹¹ Abrogating these adverse effects has become another challenge to the successful implementation of CAR T cells as part of standard therapy for relapsed/refractory ALL.

Despite the striking clinical responses achieved, and the regulatory approval of two products, it is important to keep in mind that aside from the potentially life-threatening adverse effects associated with preparations, there are also other issues associated with large-scale commercialization of this cutting-edge therapy. First, the cost of CAR T cells is much higher than the cost of CAR T-cell production. ^{1 2} Second, production that involves shipment of fresh lymphocyte preparations as well as finalized CAR T-cell products over long distances creates difficult logistical problems. These issues will undoubtedly prompt local medical (research) centers to develop their own CAR T manufacturing facilities to offer such treatment options within each country, or in several separate regions of larger countries.

Further research conducted on CAR including International Patent Publication no. WO/2018/231871 disclosing engineered T-cells, which are configured to express CAR upon reduction or knock-down of endogenous expression of a T-cell antigen. Mcginness et al. details another like research in International Patent Publication no. W02020037066 about genetically engineered hematopoietic cells, which express one or more Krebs cycle modulating polypeptides, and optionally a chimeric receptor polypeptide capable of binding to a target antigen of interest. Mcginness et al. further describes another genetically engineered hematopoietic cell, which express one or more lactate-modulating factors capable of binding to a target antigen of interest, in International Patent Publication no. W02020051493. Though both W02020037066A1 and W02020051493A1 teach about potential use of any hinge or spacer domain originated from CD28, CD16A, CD8a, or Immunoglobulin G (IgG) in assembling of a CAR capable of binding on specific antigen, but the suitable type of Immunoglobulin G (IgG) has never been identified in these disclosures.

In view of the inadequacies found in the prior research performed as discussed above, there exists a need to explore other alternatives available for acquiring a therapeutic CAR system with improved performance.

Summary

The present disclosure aims to provide a CAR configured to bind onto specific antigen expressed on a surface of a given cell type or cell lineages in deriving a beneficial outcome, particularly a therapeutic outcome, for a subject.

Further object of the present disclosure is directed to a CAR particularly designed to recognize and bind onto CD 19 expressed on the surface of B -lymphocytes or cells of B lineage applicable for a treatment of B-cell ALL. Specifically, the disclosed CAR capitalizes on modified hinge or spacer domain for obtaining an improved killing efficiency towards B -lymphocytes or cells of B lineage to treat B-cell ALL or the like illnesses.

Still another object of the present disclosure is to derive a T cell capable of expressing and presenting the disclosed CAR that binding onto the compatible antigen available on a cell surface via the CAR leads to initiation of one or more preferred immunological reactions to eradicate cancerous cells relating to B-cell lymphomas. More object of the present disclosure is to offer a viral vector incorporated with one or more polynucleotide sequences encoding for the peptides of the disclosed CAR. The disclosed vector is fashioned to express the encoded the peptides which are further assembled into the disclosed CAR in a compatible transduced cell.

At least one of the preceding objects is met, in whole or in part, by the present disclosure, in which one of the embodiments of the present disclosure is a chimeric antigen receptor (CAR) comprising an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain. The variable heavy chain region connects to the intracellular domain through a spacer, which comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1. Preferably, the spacer is Immunoglobulin (Ig) G2 -based.

For more embodiments, the variable light chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 2.

For a number of embodiments, the variable heavy chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 3.

In several embodiments, the linker comprises an amino acid sequence as setting forth in SEQ ID No. 4.

Generally, in some embodiments, the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5. Still, in more embodiments, the transmembrane domain comprises an amino acid sequence as setting forth in SEQ ID No. 6.

Preferably, in some embodiments of the disclosed CAR, the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from Oϋ3z- chain that the CD28 signaling domain and the cytoplasmic signaling domain may respectively comprises an amino acid sequence as setting forth in SEQ ID No. 7 and SEQ ID No. 8.

In more embodiments, the disclosed CAR further comprises a leader sequence precedingly connected to the variable light chain region and the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9.

Another aspect of the present disclosure refers to a genetically engineered T-cell capable of expressing a chimeric antigen receptor that the chimeric antigen receptor comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain. Preferably, the variable heavy chain region of the chimeric antigen receptor in the engineered T cell connects to the intracellular domain through a spacer, which comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.

According to several embodiments of the engineered T cell, the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5.

According to more embodiments of the engineered T cell, the intracellular domain comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from CD3ξ chain. According to a plurality of embodiments of the engineered T cell, the chimeric antigen receptor further comprises a leader sequence precedingly connected to the variable light chain region and the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9. Another aspect of the present disclosure refers to a viral vector capable of promoting expression of a peptide encoding for chimeric antigen receptor in a transduced T cell. The viral vector essentially comprises a polynucleotide sequence as setting forth in SEQ ID No. 10. Preferably, the viral vector is a lentiviral vector. Preferably, in some embodiments of the viral vector, the encoded peptide comprises at least 90% similarity of an amino acid sequence setting forth in SEQ ID No. 11.

Brief Description of The Drawings

Fig. 1 is a schematic illustration of few embodiments of the CD 19 CAR with each of the constructs comprising a CD 19 scFV, a predetermined range of IgG2 spacer, a CD28 transmembrane domain, a CD28 intracellular domain, and CD3ξ cytosolic domain;

Fig. 2 is a graph showing viable T-cell number after transduction with CAR having short spacer displayed the log phase after day 1 of transduction (similar to that of non-transduced cells) while CAR of full-spacer and intermediate-spacer entered log phase proliferation 2 days after transduction;

Fig. 3 is a graph showing percentage of transduction efficiency measured using QPCR with expression of the CAR CD28- CD3ξ domain construct in genomic DNA being detected in CD 19 CAR T cells derived from different healthy donors using QPCR with specific primers targeting the CD28- CD3ξ region, where * and # represent statistically significant differences at P < .05 when comparing with NT and the CAR full spacer construct respectively; Fig. is an Agarose gel picture after QPCR with specific primers targeting CD28-CD3ξ (the product size was 281 bp);

Fig. 5 are graphs illustrating antitumor activity of CAR T cells using a CFSE-7-AAD-based cytotoxicity assay for investigating effector to target cell ratios (E/T) in connection to different spacer length CD19 CAR T, where Raji, RS4, and Sup-B15 (CD19 expressed cells) were used as target cells, the Jurkat cell line and PHA blasts (CD 19 non-expressing cells) were used as negative controls, and non-transduced T cells (NT) were used as controls that the data is representative of three independent experiments performed with CAR T cells generated from five separate donors (*, #, and $ represent P < .05 compared with NT, Full, and Intermediate spacer length, respectively, whereas ** and *** strand for P < .01 and .001 when compared with NT);

Fig. 6 is a gel picture showing detected CD 19 CAR T cells in peripheral blood (PB) and bone marrow (BM) samples with BM being examined on day 14 of the CAR T-cell infusion (D denotes Day, + ve indicates transduced cells, and -ve represents non-transduced cells); and

Fig. 7 shows amino acid sequences of (a) SEQ ID No. 1, (b) SEQ ID No. 2, (c) SEQ ID No. 3, (d) SEQ ID No. 4, (e) SEQ ID No. 5, (f) SEQ ID No. 6, (g) SEQ ID No. 7, (h) SEQ ID No. 8, and (i) SEQ ID No. 9;

Fig. 8 shows (a) a polynucleotide sequence, denoted as SEQ ID No. 10, encoding for (b) one embodiment of a CAR, denoted as SEQ ID No. 11 , that the polynucleotide sequence can be incorporated into a viral vector. Detailed Description

Hereinafter, the disclosure shall be described according to the preferred embodiments and by referring to the accompanying description and drawings. However, it is to be understood that referring the description to the preferred embodiments of the disclosure and to the drawings is merely to facilitate discussion of the various disclosed embodiments and it is envisioned that those skilled in the art may devise various modifications without departing from the scope of the appended claim.

As used herein, the terms “approximately” or "about", in the context of concentrations of components, conditions, other measurement values, etc., means +/- 5% of the stated value, or +/- 4% of the stated value, or +/- 3% of the stated value, or +/- 2% of the stated value, or +/- 1% of the stated value, or +/- 0.5% of the stated value, or +/- 0% of the stated value.

The term "polynucleotide" or "nucleic acid" as used herein designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers to oligonucleotides greater than 30 nucleotide residues in length.

The term “chimeric antigen receptor” or “CAR” used herein throughout the specification refers to a man-made construct or complex, assembled from one or more peptides, comprising multiple domains that at least two of domains forming the construct or complex are of different origins.

One aspect of the present disclosure refers to a CAR, which may adopt various arrangements or configuration as generally illustrated in different embodiments shown in Fig. 1. Particularly, the disclosed CAR comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end- to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain. It is important to note that the disclosed CAR is CD 19 specific, which is ubiquitously expressed on the surface of B-lymphocytes or cells of B-lymphocytes lineage. Thus, an engineered T cell carrying the CD 19-specific CAR shall facilitate recognition of the cells of B-lymphocytes lineage by the engineered T cell and coupling thereof. The coupling of the engineered T cell and cell of B- lymphocyte lineage may lead to activation of the T cell and release of corresponding immunoregulatory cytokines that finally results in killing or eradication of the recognized cells of B -lymphocytes lineage. The aforesaid interaction occurred between the engineered T cell and cells of B -lymphocytes lineage enables the disclosed CAR to equip the engineered T cell the functionality to serve as a means to treat B-cell ALL, the like illness and/or any complications associated thereto. Despite the CAR described hereinafter is commonly directed to a second generation CAR, some embodiments of the disclosed CAR may include CAR of third or fourth generation

Referring again to Fig.1 , the variable heavy chain region of the disclosed CAR connects to the intracellular domain through a spacer. The spacer commonly serves to provide enough space between the antigen binding domain and the membrane plasma of the transduced cells such that the antigen binding domain can be fold intrinsically free from interruption from plasma membrane or any cellular components mounted on the membrane. Despite it was previously reported that the length of the spacer may play a role in determining efficiency of the T-cell carrying the CAR for leukemia treatment, the inventors of the present disclosure however discovered that the rightful length of the spacer in fact affect transduction efficacy, which can be another important factor to decide treatment efficiency of the engineered T cells. Particularly, the spacer of the disclosed CAR is of IgG2 origin or, more particularly, the spacer is human IgG2 origin. The spacer can be a continuous segment or region derived from human IgG2, with or without further modifications, in some other embodiments of the disclosed CAR. For more embodiments of the disclosed CAR, the spacer comprises a hinge region, a CH3 region, and a CH2 region interposing between the hinge region and the CH3 region. Moreover, it was found by inventors of the present disclosure that the employment of a spacer derived from IgG2-based hinge region, a CH2 region and a modified CH3 region having a length of around 132 amino acids may render the disclosed CAR with better clinical outcome against cancerous cells of B -lymphocyte lineage. More preferably, the spacer comprises an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.

For some embodiments, the antigen binding domain is a single-chain variable fragment. As setting out in the foregoing description, the antigen binding domain is essentially composed of a variable light chain region and a variable heavy chain region joined in tandem through a linker. In more specific, the linker employed in some of the embodiments can be flexible GS linkers essentially composed of glycine (Gly) and serine (Ser) residues. More preferably, a G4S or (Gly-Gly-Gly-Gly-Ser) _n flexible linker which is resistant against cellular protease activities is employed to fuse the variable heavy chain region and variable light chain region together, where n can range from 1 to 10. (G4S) ₄ is preferably utilized in some embodiments of the disclosed CAR to sufficiently space the light and heavy chain regions apart without causing any undesired interaction between the two separated regions yet retaining the anti-CD 19 property of the antigen binding domain resided in the extracellular domain. Therefore, in a number of the preferred embodiments, the linker comprises an amino acid sequence as setting forth in SEQ ID No. 4. The employed linker connects the amino-terminal of the variable light chain region to the carboxy- terminal of the variable heavy chain region. As to the variable light and heavy chain regions, these domains are generally derived respectively from light and heavy chains of immunoglobulin of Mus musculus, with or without further modifications depending on the embodiments of the disclosed CAR. Preferably, the variable light chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 2. Likewise, the variable heavy chain region comprises an amino acid sequence with at least 90% similarity of SEQ ID No. 3. In more particular, the extracellular domain spanning across the variable light chain region, the linker and the variable heavy chain region comprises an amino acid sequence as setting forth in SEQ ID No. 5.

According to further embodiments, the disclosed CAR may further comprise a leader sequence or leader peptide sequence that assist expression of the CAR on the engineered T cell membrane after secretion from Golgi complex. Preferably, the leader peptide sequence precedingly connects to the variable light chain region for guiding the secreted chimeric peptide towards the surface membrane for expression. The leader sequence may comprise an amino acids sequence, but not limited to, as setting forth in SEQ ID No. 9.

Still, in more embodiments of the disclosed CAR, the intracellular domain is preferably TCR zeta chain or structure configured to facilitate transfer of stimulating signal to activate T cells upon reacting of the antigen binding domain towards the CD 19 located on the cells of B -lymphocytes lineage. More specifically, the intracellular domain of the comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from human CD3ξ-chain . Preferably, the CD28 signaling domain comprises an amino acid sequence as setting forth in SEQ ID No. 7 while the cytoplasmic signaling domain comprises an amino acid sequence as setting forth in SEQ ID No. 8.

In accordance with a plurality embodiment of the disclosed CAR, the transmembrane domain comprises an amino acid sequence as setting forth in SEQ ID No. 6, which is preferably originated from human CD28 specifically found on surface of the T cells.

Pursuant to another aspect of the present disclosure, a genetically engineered T-cell capable of expressing a CAR configured to couple with CD 19 found on cellular surface of cells of B- lymphocyte lineage that binding of the CAR onto the CD 19 invokes a cascade of immunoregulatory reactions leading to killing or eradication of the bound cells of B -lymphocyte lineage. It is an effort of the inventors of the present disclosure to disclose an effective means for the treatment of B-cell-ALL via cell therapy through assembling a CD 19-specific chimeric antigen receptor for cell. Like described in the foregoing, the disclosed engineered T cell possesses better transduction efficacy and killing efficiency over the recognized or identified cells of B-lymphocyte lineage in view of the application of a spacer incorporated with modified CH3 domain of IgG2. Preferably, the disclosed engineered T cell comprises a plurality of chimeric antigen receptor, which essentially comprises an extracellular domain having an antigen binding domain to be disposed on a surface of a plasma membrane of a modified T-cell, the antigen binding domain comprising a variable light chain region and a variable heavy chain region connecting in an end-to-end fashion through a linker; an intracellular domain for regulating activation of the modified T-cell; and a transmembrane domain anchoring on the plasma membrane of the modified T-cell and interposing in between the extracellular domain and the intracellular domain. To improve transduction efficacy and killing efficiency over the targeted cells of B-lymphocyte lineage, the variable heavy chain region connects to the intracellular domain through a spacer comprising an amino acid sequence of at least 90% similarity of a sequence as setting forth in SEQ ID No. 1.

In a number of embodiments, the CAR of the disclosed engineered T cell may include a segment of leader sequence precedingly connected to the variable light chain region. Preferably, the leader sequence has an amino acids sequence as setting forth in SEQ ID No. 9 such that the secreted peptide or polypeptide of the CAR can be effectively expressed on the surface of the engineered T cells. It is crucial to note that the disclosed engineered T cells can become a tool for the treating B-cell leukemia patients by cell therapy thus resolving any complications associated with the use of standard chemotherapeutic drugs. Nonetheless, it is possible also to run a leukemia treatment using the disclosed engineered T cell along with predetermined chemotherapeutic drugs to yield the desired therapeutic outcome yet ensuring the side effects elicited by the chemotherapeutic drugs are minimal.

According to several embodiments of the disclosed engineered T cell, the extracellular domain comprises an amino acid sequence as setting forth in SEQ ID No. 5, which spanning across a variable light chain region, a linker and a variable heavy chain region respectively comprise amino acid sequence as setting forth in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4.

As described above, the intracellular domain of the CAR expressed in the disclosed engineered T-cell comprises a human CD28 signaling domain coupled to a cytoplasmic signaling domain derived from CD3ξ-chain.

Another aspect of the present disclosure relates to viral vector capable of promoting expression of a peptide, which corresponds to one or more embodiments of the disclosed CAR mentioned above. Particularly, the disclosed viral vector is encoding for a chimeric antigen receptor which can be expressed in a compatible transduced T cell under a predetermined culturing condition. The disclosed viral vector comprises a polynucleotide sequence or a base sequence as setting forth in SEQ ID No. 10. It is possible to further modify the encoding sequence of SEQ ID No. 10 to derive a CAR with improved or altered properties for the treatment of B-cell ALL, the like illness and/or any complications associated thereto. As such, more embodiments of the disclosed viral vector comprise a polynucleotide sequence or a base sequence of at least 90% similarity as of setting forth in SEQ ID No. 10. The disclosed viral vector can be employed for the creation of the CD 19-specific chimeric-antigen-receptor T-cells, which in turn being applicable as a treatment for B-cell leukemia patients through any established protocol for leukemia cell therapy. Furthermore, the peptide being encoded by the disclosed vector may comprise at least 90% similarity of an amino acid sequence as setting forth in SEQ ID No. 11. Preferably, the disclosed viral vector is a lentiviral vector.

Again, one aspect of the present disclosure is associated to a genetically engineered T-cell as disclosed or described above for use as a medicament in a treatment of B-cell ALL.

The following example is intended to further illustrate the disclosure, without any intent for the disclosure to be limited to the specific embodiments described therein.

Example 1

The two CAR constructs, CAR-CD19-TM28-Z and CAR-PSCA- CD28TM-CD28- CD3ξ, were kindly provided by the Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, and Houston Methodist Hospital, Houston, TX, USA. They were ligated to produce second-generation anti-CD19 scFv-CD28- CD3ξ. Modified CAR constructs with different spacer lengths, “intermediate” and “short,” were made using anti-CD 19 scFv-CD28- CD3ξ as a template. The intermediate and short constructs had CH2 and CH2CH3 deletion, respectively. The three CAR constructs were subcloned into a lentiviral vector backbone plasmid (pSin-EF2-Puro; Addgene, Watertown, MA, USA).

Example 2

HEK 293T cells (5 x 10 ⁶ cells per 10-cm dish) were plated into poly-D lysine (Sigma-Aldrich, St. Louis, MO, USA)-coated plates prior to co-transfection of envelope (pMD2.G) and packaging (psPAX2) plasmids with pSIN-CD19 CAR vector. On the following day, HEK 293T cells were transfected using FuGENE HD Transfection Reagent (Promega, Madison, WI, USA). Supernatants were collected 48 and 72 h post-transfection and filtered through 0.45-mih polyethersulfone filter (Merck Millipore, Burlington, MA, USA) to remove cell debris. Lenti viruses were concentrated using Lenti-X Concentrator (Takara, Shiga, Japan) according to the manufacturer’s instructions. Briefly, the supernatants were mixed with Lenti-X Concentrator at a ratio 1:3 (Lenti-X Concentrator: supernatant) and the mixtures were incubated for 30 min at 4°C. Thereafter, the mixtures were centrifuged at 1500 x g for 45 min at 4°C. The pellet was suspended in Dulbecco’s phosphate-buffered saline (DPBS) (GIBCO; Thermo Fisher Scientific, Inc, Waltham, MA, USA) and stored at -80°C until use.

Example 3

Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors and were isolated by density gradient centrifugation using Ficoll-Paque (GE Healthcare Bio-Sciences Corp, Piscataway, NJ, USA). The experiment was approved by the Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (pro- tocol ID 03-59-11). The PBMCs were then cultured in TexMACS GMP medium (MACS; Miltenyi Biotec) with 5% heat-inactivated human AB serum (Sigma- Aldrich Pte Ltd) and recombinant human IL2200 U (GMP grade, CellGenix; CellGenix Inc). The Raji cell line (Burkitt’s lymphoma) was kindly provided by Assistant Professor Dachrit Nilubol. The Jurkat cell line (acute T- lymphoblastic leukemia) was kindly provided by Professor Kovit Pattanapanyasat. RS4 and Sup-B15 cell lines (acute lymphoblastic leukemia) were purchased from American Type Culture Collection (Manassas, VA, USA). The Raji, RS4, and Jurkat cell lines were cultured in RPMI- 1640 medium with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin/streptomycin (GIBCO), whereas Sup-B15 was cultured in Iscove’s modified Dulbecco’s medium (Hyclone, GE Healthcare Life Sciences) with 20% FBS, 1% penicillin/streptomycin, and 0.05 mM 2- mercaptoethanol (Sigma- Aldrich).

For the generation of allogeneic phytohaemagglutinin-stimulated (PHA) blast cells, 5 x 10 ⁵ PBMCs were stimulated with 5 μg/mL PHA (Invitrogen; Thermo Fisher Scientific, Inc) in RPMI- 1640 medium with 10% FBS and 1% penicillin/streptomycin. A total of 100 U/mL IL-2 (PeproTech, Rocky Hill, NJ, USA) was added on day 2. PHA blast cells were cultured for 7 days. The medium and IL-2 were replaced every 2- 3 days. All cell cultures were maintained at 37°C in a fully humidified atmosphere of 5% CO2 in air.

Example 4 T cells were stimulated, using antihuman CD3 (MACS; Miltenyi Biotec) and anti-CD28 (MACS; Miltenyi Biotec) monoclonal antibodies, and then cultured in TexMACS GMP medium with 5% heat-inactivated human AB serum and recombinant human IL2200 U (GMP grade). Transduction was carried out on day 3. Activated T cells were transduced using RetroNectin (Takara, Shiga, Japan). RetroNectin was used at a concentration of 20 μg/mL in DPBS to coat the 24-well plate. The plate was incubated for 2 h at room temperature and blocked with 2% heat-inactivated human AB serum (Sigma- Aldrich) in DPBS. The plate was then washed once with DPBS. Lentiviruses were added into 500μL complete TexMACS GMP medium and added into the coated well. The coated plate containing viruses was centrifuged at 2000 x g at 32°C for 1.5 h. Thereafter, 105 activated T cells in 500μL complete TexMACS GMP medium with IL-2 at a final concentration of 100 U/mL were added to each well. The complete medium and IL-2 were replaced every 2-3 days. Transduced T cells were cultured for 6 days for cytotoxicity assay. Example 5

DNA was extracted from transduced CD 19 CAR T cells and non-transduced T cells using GenUP gDNA Kit (Biotechrabbit, Hennigsdorf, Ger- many). Quantitative polymerase chain reaction (QPCR) was performed to determine the copy number of transgene, using QPCR Green Master Mix LRox (Biotechrabbit), and it was performed with the CLX96 Real-Time PCR Detection System (Bio-Rad, Singapore), according to the manufacturer’s instructions. The present disclosure used primers that were specific for the CD28CD3ξ domain, such that the PCR would display the same product regardless if the DNA came from cells transduced with a full-, intermediate-, or short-length spacer domain. Each reaction contained 100 ng of genomic DNA, and each experiment was performed in triplicate. The CAR copy number quantification was based on a comparison with a standard curve. Genomic DNA of non-transduced T cells was spiked with plasmid containing transgene in a serial dilution to obtain a standard curve.

To investigate the effectiveness of antiCD 19 -targeting CARs, all constructs in the present disclosure including scFv, the transmembrane, and signaling domains were identical, each encoding a CD28 transmembrane, CD28 signaling domain, and a CD3ξ-derived signaling domain (Fig. 1). Proliferation of T cells was detected after transduction. The lentiviral transduction process did not show any adverse effects on the growth and viability of the T cells. CAR full and intermediate spacer constructs entered log phase growth 2 days after the transduction, whereas the CAR short spacer construct entered log phase proliferation within 24 h after the transduction (Fig. 2). CAR construct expression was measured by QPCR as an indicator for transduction efficiency. The CAR copy number of all constructs was calculated before co-culture with target cells. All constructs expressed different transduction efficiency with approximately 13%, 41%, or 21% for full, intermediate, or short spacer, respectively (Fig. 3). The QPCR products of genomic DNA from CAR T cells and non-transduced T cells (NT) cells were subjected to agarose gel electrophoresis. The size of the respective CAR products was about 281 bp, whereas, as expected, the NT control cells did not express the CAR construct as shown in Fig. 4.

Example 6

The cytotoxicity assay was performed using flow cytometry -based analysis. Effector cells (T cells) were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen; Thermo Fisher Scien- tific, Inc) at a concentration of 1 mM. Effector cells were co-cultured with all target cells at ratios of 1:1, 10:1, and 20:1 (Effector: Target) overnight. Control samples, which only had target cells, were used to assess spontaneous cell death. On the following day, cells were collected and stained with 5 μL of 7-AAD (7-amino-actinomycin D) (eBioscience; Thermo Fisher Scientific, Inc). Data analysis was carried out by BD FACSVerse flow cytometry (BD Bioscience; San Jose, CA) with BD FACSuite software (BD Bioscience). The target cells were gated as the CFSE-negative cell population. The target cell death was determined as the CFSE negative with 7-AAD-positive cell population. The percentage of cytotoxic activity was calculated based on the following equation: Cytotoxicity (%) = (Target cell death - spontaneous cell death) x 100 100 - Spontaneous cell death

Example 7

The present disclosure monitored CD 19 CAR T cells in peripheral blood and bone marrow specimens by RT-PCR as described in Section 2.5 by using the same primers specific for the CD28CD3ξ domain.

Briefly, 2 million cells from bone marrow aspiration specimens are incubated with a premixed antibody combination for 15 min at room temperature. Then, red blood cell lysis was done by adding a lysis buffer and incubating for 10 min. All cells are washed twice and resuspended in 0.5% paraformaldehyde for acquisition by using BD LSRFortessa (Beckton Dickinson, San Jose, CA, USA). The minimal residual disease (MRD) panel is a 14-color panel with 27 leukemia- associated markers including CD37, CD164, CD58, CD304, CD20, CD81, CD200, CD24, CD10, CD38, CD44, CD79b, CD49f, CD10, CD123, CD73, CD13, CD33, CD15, CD86, CD66c, NG2(7.1), CD72, CD19, CD10, CD34, and CD45. For analysis, after doublet discrimination, immature B cells are gated by using CD 19, CD79a, CD 10, CD34, CD45, FSC, and SSC. An immunophenotypic pattern of gated immature B cells is compared to the template of hematogones. The residual mature B cells are used as an internal control. MRD is considered as a positive result when there are two or more aberrancies compared with the normal template and MRD pattern at diagnosis. The lower limit of detection is 0.01% of all nucleated cells.

To verify the increased cytotoxicity of CD 19 CAR T cells, cytotoxicity experiments were performed with three B cell AFF cell lines: Raji, RS4, and Sup-B15. The specific cytotoxicity of unsorted CD 19 CAR T cells against lymphoma cells was evaluated with the flow cytometry- based assay. The effector cells were CD 19 CAR T cells of three different constructs — full, intermediate, and short length, as shown in Figure 1. NT served as negative control T cells. All three constructs of CAR T cells exhibited significantly increased cytotoxicity against all B-cell lines when compared with NT (35-50% vs <10%). Different target cells showed different response to CD 19 CAR T cells. All constructs expressed a high level of killing of Raj i cells when compared with NT (Figure 5A), whereas short spacer CARs displayed the highest level of cytotoxicity of the RS4 (Figure 5B) and Sup-B15 (Figure 5C) cell lines. In contrast, there was no substantial difference in tumor killing ability between NT and all construct of CAR T cells against Jurkat (Figure 5D) and PHA blast cells (Figure 5E), indicating that CAR T cells were CD 19 specific.

In recent years, several clinical trials have showed impressive results from CD 19 CAR T-cell therapy in patients with B-cell malignancies. ^17-20 Nevertheless, the development of CAR design is an ongoing process in order to optimize clinical efficacy and minimize clinical toxicity of such treatment. Different costimulatory molecules such as CD28, 4- IBB, and 0X40 have been utilized to test CAR T cell efficacy against tumor cells. Other structural units, such as the CH2CH3 spacer domain, have been reported to play a role in persistence and survival of CAR T cells in vivo. ² Even though different constructs have significant differences in the costimulatory domain, such alterations may contribute to observed differences in overall therapeutic efficacy in patients with various B-cell malignancies, as well as differences in the CAR T construct’s ability to elicit varying degrees of clinical adverse events.

Several investigators ^7,21-25 have demonstrated that the spacer length is a critical issue for CAR T- cell efficiency. In this study, the present disclosure investigated a second-generation anti-CD 19 CAR T cells containing three different lengths of the spacer region; full length (CH2CH3 spacer), “intermediate” (-/CH3 spacer), and “short” (-/- spacer). The present disclosure expressed all three different lengths of the spacer region CAR constructs on the T cells, with intermediate length showing the highest transduction efficiency.

The antitumor efficacy and CD 19-specific activity levels were comparable across all three CAR constructs. We assessed the in vitro killing activities of the T cells expressing each construct against three different target cell lines with positive CD19 expression (Raji, RS4, and Sup- B15) and against one CD 19-negative cell line (Jurkat) as a control. The present disclosure found that CD 19 CAR T cells with full, intermediate, or short spacers did not have a higher percentage of specific Jurkat cell lysis when compared with that of NT, whereas all three types of CD 19 CAR T cells had higher percentages of specific lysis of Raji, RS4, and Sup-B 15 cells at an E:T ratio of 10:1 as compared with the killing effect of NT. Differences in the killing effects of the full, intermediate, and short CD19 CAR T cells were clearly observed at an E:T ratio of 20:1. Thus, modified T cells with any of the three constructs presented CD 19-specific activity. Our results are similar to those previously reported by Almåsbak et al., ⁴ which revealed that CD 19 CAR T cells expressing CAR constructs with or without the CH2CH3 spacer had specific activity in vitro but lacked antileukemia activity in vivo with partly CD 19 -independent toxicity relating to the IgGl -derived CH2CH3 spacer. In the absence of a CH2- domain, CAR constructs eliminate leukemia in vivo, without dominant toxicity.

Interestingly, although the killing effects across all three CAR constructs were comparable, the short CD 19 CAR T cells displayed the highest killing efficiency against all CD 19-expressing target cells despite its transduction efficiency being less than that of the intermediate CD 19 CAR T cells. For Raji cells, the full and intermediate CD 19 CAR T cells each displayed a fraction of specific lysis that was similar to that of the short CD 19 CAR T cells. However, their killing efficiencies against the RS4 and Sup-B 15 cells, which express relatively less CD 19 on their cell surfaces, were lower than their killing efficiencies against the Raji cells. The difference in CAR T-cell killing potency could be an effect of the presence of target antigens and the intensity of antigen expression. ²⁶ However, the modified T cells expressing any of the three constructs had higher efficiencies for eradicating CD 19-expressing cells compared with NTs. We also observed that the modified CAR T cells expressing any of the three constructs did not kill allogeneic PHA blasts. Thus, the disclosed CAR-expressing T cells appear to be specific against CD 19-expressing cells and have minimal killing effects on CD 19-negative allogeneic cells.

The CRS following CAR T-cell infusion is one of the most severe toxicities, ^27-29 which is correlated with the progressive immune activation. ^26,30 Ravanpay et al. have engineered a second- generation CAR construct with three spacer variants: full, intermediate, and short. They reported that all three spacer variants had the same tumor lytic activity. The short spacer CAR T cells induced the most robust effector cytokine production — interleukin-2, interferon-gamma, and tumor necrotic factor alpha levels, ³¹ whereas full spacer CAR T cell lacks in vivo efficiency due to the engagement of FcγR on mouse macrophages. ^4,22 Therefore, the present disclosure chose the intermediate CD 19 CAR T cells to avoid CRS. Example 9

An 11 -year-old boy with B-cell ALL had bone marrow relapse before enrollment in the compassionate clinical CAR CD 19 T-cell protocol. He was diagnosed with B-cell ALL (initial white blood cell count 1.3 x 10 ⁶ cells/μL) 8 years earlier. His cytogenetic study revealed 46, XY. He was treated with the standard risk ThaiPOG protocol. ¹³ Two years later, he developed bone marrow relapse, and he was treated with the relapsed ALL protocol (modified SJCRH relapse

ALL protocol). ¹⁴ One month after he finished treatment on the modified SJCRH protocol, he suffered a second bone marrow relapse. He subsequently received therapy on a relapse ALL protocol following the UK ALL regimen. ¹⁵ After he had achieved CR, he underwent haploidentical HSCT from his mother. One year later, he had a central nervous system (CNS) relapse with an intracranial mass. He achieved a new CR with a bortezomib- based regimen ¹⁶ and was also given intrathecal chemotherapy and craniospinal irradiation. Six months after finishing treatment for his CNS involvement, he again had a systemic bone marrow relapse with 30% blasts. Reinduction chemotherapy consisted of a combination of bortezomib, cytarabine, and etoposide, ¹⁶ and under a compassionate investigation new drug (IND) he was consolidated with haploidentical CD19 CAR T cells at a dose of 0.52 x 10 ⁶ cells/kg body weight (0.8 x 10 ⁶ total T cells/kg; 65% transfection efficiency intermediate length spacer construct). The only side effect experienced after the CAR T-cell treatment was low-grade fever, but without clinical signs of graft versus host disease or serious CRS, so it was decided to only observe and closely follow the patient till his low-grade fever subsided. By day 30 after the CAR T-cell infusion, he was documented to have achieved a MRD-negative CR as shown in Table 1, which has been continuing for 10+ months.

Mote: Day0= day of CD19CAR19 T-cells infusion

Table 1

In reference to the patient with B-cell ALL, inventors of the present disclosure gave him one cycle of chemotherapy prior to CAR CD 19 T-cell treatment to reduce the leukemic blasts and induce an immunosuppressed state, conducive to the haploidentical CAR T-cell infusion. He then received haploidentical CD 19 CAR T cells under a compassionate use plea. The present disclosure demonstrated induction of CR and also the continuous, unmaintained remission of refractory leukemia that remains MRD negative for 10+ months. Genetically modified CD19 CAR T cells were detected in both peripheral blood and bone marrow samples by RT-PCR for at least 2 weeks after the CD19 CAR T-cell infusion (Fig. 6). With these favorable findings, the present disclosure planned to investigate allogeneic CD 19 CAR T cells in relapsed/refractory B- cell ALL patients in a phase I-II setting as bridging therapy to allo-HSCT, followed by a posttransplant CAR T-cell boost as consolidation in these high-risk patients, because the present disclosure was concerned about the possible deleterious effect of the pretransplant conditioning chemotherapy on the CAR T population that was administered as salvage therapy prior to HSCT.

It is to be understood that the present disclosure may be embodied in other specific forms and is not limited to the sole embodiment described above. However, modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto. References

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