DESC:
FIELD OF THE INVENTION
The present invention relates to novel chimeric CD20-targeting Chimeric Antigen Receptor (CAR) consisting of anti-CD20 binding domain effective for treatment of patients suffering from cancer involving B cells, such as Acute Lymphoblastic Leukemia (ALL) and B cell Non-Hodgkin's Lymphoma (B-NHL) and other cancers, autoimmune diseases, non-autoimmune inflammatory diseases, transplantation and other B cell-related disorders.

BACKGROUND OF THE INVENTION
Leukemia is one of the common cancers in the world. Although remarkable cure-rates (over 90% in US and Europe) have been observed in childhood Acute Lymphoblastic Leukemia (ALL) over last few decades, much less success has been observed in adult ALL (<50%) and other forms of leukemia. In India, the limited epidemiology register-based data sets indicate lower cure-rates in children (~60%-75%) and even lower in adults. Allogeneic stem cell transplant (SCT) therapy has been successfully used for treatment of relapsed-ALL subjects; however, this treatment is available only to a small subset of patients, since patients must be in remission before the SCT treatment.
Thus, there is a serious unmet need in India to treat large numbers of patients with ALL and B-NHL with innovative therapies. Recent successes in Chimeric Antigen Receptor T Cell therapy (CAR- T) for patients that have failed conventional therapy, offers such an opportunity. CAR-T cell therapy is a form of cell therapy wherein a patient or to express a receptor that enables them to kill the tumor cells (US7638325). This process was approved against leukemia wherein a B cell marker CD19 was targeted by CAR-T cells (US8911993). However, there have been cases of antigen escape, wherein CD19 expression is lost by the B cells rendering the CAR-T therapy to be ineffective.

US2020002400 discloses chimeric antigen receptor targeting CD20 antigen and a preparation method thereof. The extracellular antigen binding domain of the chimeric antigen receptor includes an antibody heavy chain variable region and an antibody light chain variable region, and is capable of killing tumor cells. US2020002400 uses a CD8 leader signal sequence that enables the extracellular export of the CAR. The present inventors have advantageously used a different leader sequence, a native leader sequence of the heavy chain of the scFv that enhances the export of the CAR to the surface. An enhanced export of the CARs to the surface by the specific leader/ signal sequence leads to higher number of CARs being expressed on the T cells in the present invention. A higher number of CARs on the cells translates into better efficacy (cytotoxicity and cytokine release).

WO2020061048 discloses nucleic acids comprising a nucleotide sequence encoding chimeric antigen receptor (CAR) amino acid constructs. Polypeptides, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions relating to the CAR constructs are disclosed. WO2020061048 also discloses methods of detecting the presence of cancer in a mammal and methods of treating or preventing cancer in a mammal. WO2020061048 relates to bispecific chimeric antigen receptor. A bispecific CAR sometimes leads to a severe cytokine release syndrome due to uncontrolled cytolytic activity caused due to the bispecific targeting. A monospecific CAR on the other hand is tuned to release cytokines that does not lead to excess release of cytokines. WO2020061048 discloses IFN-? release in the range of 1300 – 14300 pg/ml and IL-2 release in the range of 16 – 215 pg/ml. IFN-? is known to have an anti-tumor activity; however, an excess of IFN-? plays a role in cytokine release syndrome wherein it causes more damage to healthy tissue than only kill tumor cells. Thus, there is a need to provide appropriate release of IFN-? where tumor related cytotoxicity is observed but minimal non-tumor related toxicity as in the present invention. IL-2 is a cytokine that plays a major role in T cell proliferation. IL-2 is released by the CAR-T cells and has an autocrine effect wherein it aids in proliferation of these cells. Thus, a higher expression of IL-2 leads to better cytotoxicity against tumor cells. The present inventors have advantageously developed a CAR construct wherein the IFN-? release is low of about 400 – 600 pg/ml and high IL-2 release of about 2000-4000 pg/ml respectively.

US9382327 discloses compositions and methods for treating diseases associated with CD20, including lymphomas, autoimmune diseases, and transplant rejections. Compositions include anti-CD20 antibodies capable of binding to a human CD20 antigen located on the surface of a human CD20-expressing cell, wherein the antibody has increased complement-dependent cell-mediated cytotoxicity (CDC) that is achieved by having at least one optimized CDR engineered within the variable region of the antibody. Compositions also include antigen-binding fragments, variants, and derivatives of the monoclonal antibodies, cell lines producing these antibody compositions, and isolated nucleic acid molecules encoding the amino acid sequences of the antibodies. The invention further includes pharmaceutical compositions comprising the anti-CD20 antibodies of the invention, or antigen-binding fragments, variants, or derivatives thereof, in a pharmaceutically acceptable carrier, and methods of use of these anti-CD20 antibodies.

US9382327 discloses use of antibodies against CD20 to treat respective malignancies. The present invention on the other hand relates to chimeric antigen receptor and not an antibody. While both the elements (antibody and CAR) invoke an immune response against target cells, a CAR leads to exclusive activation of T cells targeted against the tumor while an antibody leads to activation of other immune cells like the NK cells, macrophages, etc., to elicit an immune response.

Further an antibody is a small molecule with a limited half-life and cannot multiply while CAR-T cells expressing the CAR are cells that multiply and lead to continued immune response. Antibodies have to be administered repeatedly to control the disease while CAR-T cells expressing the CARs have to be administered only ones thus having better safety. Further some of the antibodies disclosed in US9382327 are mouse antibodies which may be rejected thus reducing efficacy.

WO2019126724 discloses a chimeric antigen receptor (CAR) comprising: a) a single chain Fv antibody (scFv) that binds a first antigen; b) a single domain antibody (sdAb) or antigen binding fragment thereof that binds a second antigen; b) a transmembrane domain; c) one or more intracellular costimulatory signaling domains; and/or d) a primary signaling domain. WO2019126724 relates to bispecific chimeric antigen receptor. A bispecific CAR sometimes leads to a severe cytokine release syndrome due to uncontrolled cytolytic activity caused due to the bispecific targeting. A monospecific CAR on the other hand is tuned to release cytokines that does not lead to excess release of cytokines. Table A below shows the comparative release profile of biphasic CAR of WO2019126724 vis-à-vis that of the present invention.

Table A
IFN-? TNF-A IL-2
Present Invention 400 – 600 pg/ml 600pg/ml 2000-4000 pg/ml
WO2019126724A1 5000 – 8000 pg/ml 600 pg/ml 50 – 500 pg/ml

The present invention provides a CAR-T cell therapy targeting CD20, which is another B cell marker. Rituximab, a monoclonal antibody targeting CD20 has proven to be successful in the clinics; hence the inventors of the present invention have targeted CD20 in a CAR-T format for use in treatment of patients suffering from cancer involving B cells such as Acute Lymphoblastic Leukemia (ALL) and B cell Non-Hodgkin's Lymphoma (B-NHL) and other cancers and autoimmune diseases, non-autoimmune inflammatory diseases, transplantation and other B cell-related disorders.

OBJECT OF THE PRESENT INVENTION
It is an object of the present invention to overcome the drawbacks of the prior arts.

It is another object of the present invention to provide a Chimeric Antigen Receptor (CAR) consisting of anti-CD20 binding domain effective for treating patients suffering from a disease or disorder selected from a group consisting of cancer involving B cells, Acute Lymphoblastic Leukemia (ALL) or B cell Non-Hodgkin's Lymphoma (B- NHL), autoimmune disease, non-autoimmune inflammatory disease, transplantation and B cell-related disorder.

SUMMARY OF THE PRESENT INVENTION
Accordingly, the present invention provides a polynucleotide encoding a chimeric antigen receptor (CAR) construct comprising:
a) a signal peptide
b) an antigen binding domain;
c) a hinge region;
d) a transmembrane domain;
e) one or more co-stimulatory domain;
f) a signaling domain,
said antigen binding domain being selected from a CD20 binding domain (scFv) or an anti - CD20 binding domain (scFv);
said scFv comprising a heavy and a light chain in a format of heavy-light, light-heavy or heavy-heavy chains;
said signal peptide having the sequence SEQ ID 24.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
The above and other aspects, features and advantages of the embodiments of the present disclosure will be more apparent in the following description taken in conjunction with the accompanying drawings, in which:

Figure 1 illustrates the plasmid map for mRNA expression by in-vitro Transcription (IVT) for designing and synthesis of plasmid DNA constructs for CAR expression in T cells.

Figure 2 illustrates agarose gel electrophoresis of PCR amplified pmRNAxp Plasmid backbone.

Figures 3 and 4 illustrates the results of the PCR amplification of the respective Chimeric Antigen Receptor (CAR) gene inserts.

Figure 5 illustrates the immunophenotyping of donor NHHV-17166 72hr post stimulation with CD3/CD28 beads.

Figure 6 illustrates the spontaneous cytotoxicity of target cells from donor NHHV-17166.

Figure 7 illustrates the assessment of T cell purity post isolation and before transfection.

Figure 8 illustrates the protein L staining for donor NHHV-17166 (untransfected).

Figure 9 illustrates the Protein L staining after CD20 CAR mRNA transfections.

Figure 10 illustrates the Target cell profiling considered for cytotoxicity assays; indicating the surface staining of CD19 and CD20 receptors on B cell lines with monoclonal antibodies against CD19 and CD20.

Figure 11 illustrates data summarizing cytotoxicity response of CD20 CART cells performed in DAUDI, RAJI and K562 cells at 48h.

Figures 12A and 12B illustrates the Effector/stimulator cytokines and Inflammatory/Regulatory cytokines release by CD20 CAR-T cells on co-culture with DAUDI cells and RAJI cells respectively.

Figures 13A-13C illustrates the cytokine analysis for DAUDI cells, RAJI cells and K562 cells respectively.

Figure 14 illustrates data summarizing levels of all cytokine analysis for DAUDI, RAJI and K562 cells.

Figures 15A-15C illustrates Cell proliferation assay considering DAUDI, RAJI and K562 cells respectively for both test and control samples.

DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a novel polynucleotide construct encoding Chimeric antigen receptor (CAR) comprising CD20 binding domain and its use in CAR-T cell therapy for treatment of patients suffering from cancer involving B cells such as Acute Lymphoblastic Leukemia (ALL) and B cell Non-Lymphoma (B-NHL) and other cancers and autoimmune diseases, non- autoimmune inflammatory diseases, transplantation and other B cell-related disorders.

The following description with reference to the accompanying drawings is provided to assist in a comprehensive understanding of exemplary embodiments of the invention. It includes various specific details to assist in that understanding but these are to be regarded as merely exemplary. Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.

The terms and words used in the following description and claims are not limited to the bibliographical meanings, but, are merely used by the inventor to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention as defined by the appended claims and their equivalents.

It is to be understood that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. Also, expressions such as "at least one of", when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

A typical chimeric antigen receptor (CAR) construct consists of the following:

Signal peptide-Heavy/Light chain-Linker-Heavy/Light chain -Hinge-Transmembrane domain - Costimulatory domain 1- costimulatory domain 2 - activation or signaling domain.

The present invention provides a polynucleotide encoding chimeric antigen receptor (CAR) construct which comprises:

Signal peptide - CD20 binding domain (scFv)-(hinge and transmembrane region) - CD28 cytoplasmic domain (Co-stimulatory signal1) - 4-1BB cytoplasmic domain (Co-stimulatory signal 2) -CD3 zeta cytoplasmic domain (signaling domain).

In another variation, the present invention also provides a chimeric antigen receptor (CAR) construct comprising:

Signal peptide-anti CD20 scFv-CD28 (hinge and transmembrane region) - CD28 cytoplasmic domain (Co-stimulatory signal 1) - 4-1BB cytoplasmic domain (Co-stimulatory signal 2) - CD3 zeta cytoplasmic domain (signaling domain);

The present invention also provides the following variations of the chimeric antigen receptor (CAR) construct and further includes such variations that can be deduced by a person skilled in the art:

- Signal peptide - anti CD20 scFv - CD28 (hinge and transmembrane region) - 4-1BB cytoplasmic domain (Co-stimulatory signal 1) - CD3 zeta cytoplasmic domain
- Signal peptide - anti CD20 scFv - CD28 cytoplasmic domain (Co-stimulatory signal 1) - 4-1BB cytoplasmic domain (Co- stimulatory signal 2) - CD3 zeta cytoplasmic domain (signaling domain)
- Signal peptide - anti CD20 scFv - CD28 cytoplasmic domain (Co-stimulatory signal 1) - CD3 zeta cytoplasmic domain (signaling domain)
- Signal peptide - anti CD20 scFv - 4-1BB cytoplasmic domain (Costimulatory signal 2) - CD3 zeta cytoplasmic domain (signaling domain)
- Signal peptide- anti CD20 scFv - CD28(hinge and transmembrane region) - CD28 cytoplasmic domain (Co-stimulatory signal 1)- 4-1BB cytoplasmic domain (Co-stimulatory signal 2) - CD3 zeta cytoplasmic domain (signaling domain)
- Signal peptide-anti CD20 scFv - CD28 (hinge and transmembrane region) - CD28 cytoplasmic domain (Co-stimulatory signal 1) - CD3 zeta cytoplasmic domain (signaling domain)

The said CD20-targeting CAR can be constructed on the KIR platform as an alternative to other co-stimulatory domains like CD28, 4-1BB, OX40, etc.

In one embodiment of the present invention, the 'Heavy/Light Linker Heavy/Light' region is the scFv (single chain fragment variable) and consists of a heavy and light chain from a single antibody and can be in any of the following combinations/format in a CAR construct:

· Heavy-light; light-heavy; or heavy-heavy format.

The scFv is selected from mouse, human, chimeric, humanized, guinea pig, dog, llama, or camel. The heavy and light chains from different antibodies cannot be combined in one scFv. The scFv can be either mouse, human, chimeric or humanized in origin.

According to the present invention the CD20 binding domain comprises complementarity determining region (CDR) from human and mouse and framework region of human origin. The CDRs which are generally a combination of CDR1, CDR2 and CDR3, provide specificity for the target antigen forming a hypervariable region recognizing the target antigen.

According to one of the embodiments of the present invention, the polynucleotide encoding a chimeric antigen receptor comprises single chain fragment variable (scFv) or the antibody fragment comprising a murine anti-CD20 binding domain having heavy chain nucleotide sequence of SEQ ID NO 1 and light chain nucleotide sequence of SEQ ID NO 3.

According to another embodiment of the present invention, the polynucleotide encoding a chimeric antigen receptor comprises antigen binding domain from single chain fragment variable (scFv) or the antibody fragment comprising a murine anti-CD20 binding domain having heavy chain amino acid sequence of SEQ ID NO 2 and light chain amino acid sequence of SEQ ID NO 4.

According to a further embodiment of the present invention the polynucleotide encoding a chimeric antigen receptor comprises a single chain fragment variable (scFv) or the antibody fragment comprising a human-mouse chimera anti-CD20 binding domain having heavy chain nucleotide sequence of SEQ ID NO 5 and light chain nucleotide sequence of SEQ ID NO 7.

According to a yet another embodiment of the present invention, the polynucleotide encoding a chimeric antigen receptor comprises antigen binding domain from single chain fragment variable (scFv) or the antibody fragment comprising a human-mouse chimera anti-CD20 binding domain having heavy chain amino acid sequence of SEQ ID NO 6 and light chain amino acid sequence of SEQ ID NO 8.

While CD20 binding domain is used in the present invention, it is envisaged that various other B cells antigens can be targeted using CAR T cells which include but not limited to CD19, CD22, CD79b, CD38, CD27, CD138, CD123, CD70, CD30 and BCMA.

In another aspect, the polynucleotide encoding CAR construct comprising a CD20 antigen binding domain is connected to a transmembrane region via a hinge region. The transmembrane and hinge regions can be from the members of the Immunoglobulin superfamily, for example, IgA, IgD, IgE, IgG, IgM. In a preferred embodiment, the transmembrane and hinge regions can be from the members of the Cluster of Domain (CD) receptors expressed on T cells such as CD2, CD3, CD4, CD8, CD28, CD45R). The hinge and transmembrane regions can be derived from human or murine. In a more preferred embodiment, the CAR construct comprises the hinge and transmembrane regions derived from human represented by nucleotide sequence of SEQ ID NO. 9 or polypeptide sequence of SEQ ID NO. 10.

The polynucleotide encoding CAR construct can also comprise the hinge and transmembrane region derived from human CD28 represented by nucleotide sequence of SEQ ID NO. 11 or polypeptide sequence of SEQ ID NO. 12.

In another embodiment, the polynucleotide encoding CAR construct of the present invention further comprises one or more co-stimulatory domains selected from the group consisting of CD27, CD28, 4-1BB, ICOS, OX40, CD27, CD30, CD40, CD40L, PD-1, KIR-DAP12 and TLRs. These co-stimulatory signals are of human or murine origin. In a preferred embodiment, the present invention consists of CD28 cytoplasmic domain followed by 4-1BB cytoplasmic domain as shown below:

Co-stimulatory signal 1 CD28 cytoplasmic domain - Co-stimulatory signal 2 4-1BB cytoplasmic domain, wherein, Co-stimulatory signal 1, CD28 cytoplasmic domain is represented by nucleotide sequence represented by SEQ ID NO. 13 and polypeptide sequence of SEQ ID NO. 14; and Co-stimulatory signal 2,4-1BB is represented by nucleotide sequence SEQ ID NO. 15 and polypeptide sequence of SEQ ID NO. 16.

The CAR construct of the invention further provides an activation or signaling domain from CD3 zeta receptor represented by nucleotide sequence SEQ ID NO. 17 and polypeptide sequence of SEQ ID NO. 18.

According to a preferred embodiment of the present invention, the polynucleotide encoding chimeric antigen receptor targeting CD20 comprises amino acid sequence of SEQ ID NO: 20 or 22 and nucleotide sequence of SEQ ID NO: 19 or 21.

The signal peptide of the CAR construct encoded by the polynucleotide of the present invention can be any peptide fragment that can express the receptor on the cell surface. In a preferred embodiment the signal peptide has the amino acid sequence of SEQ ID NO: 23 and nucleotide sequence of SEQ ID NO: 24.

It has been surprisingly found that the signal peptide sequence having SEQ ID 23 when used with the scFv sequence selected from Sequence ID 1-8 gives a better expression of the CAR on the cell surface leading to better cytotoxicity. The CAR construct of the present invention gives a higher expression of IL-2 leads to better cytotoxicity against tumor cells and low expression of IFN-? which leads to less damage to healthy tissue. The present inventors have advantageously developed a CAR construct which shows a better effector cytokine profile and a lessor inflammatory cytokine profile, making it potentially more efficacious and lesser toxic as a therapy. The CAR construct of the present invention has IL-2 release which is about 5 to 10 times that of IFN-?. The IFN-? release is about 400 - 600 pg/ml while the IL-2 release about 2000-4000 pg/ml respectively.

The polynucleotide encoding CAR construct of the invention comprises a linker between the heavy and the light chain (or the heavy and the heavy chain) scFv fragments. The linker is typically a G3S linker represented by GGGS and its length can be in the multiples of 2, 3, 4 or 5. In a preferred embodiment the linker has the amino acid sequence of SEQ ID NO: 25 and nucleotide sequence of SEQ ID NO: 26.

In yet another embodiment the nucleotide and polypeptide sequences disclosed herein further include variants, derivatives, fragments; preferably the invention encompasses modifications to the extent of 95% homology/identity to the disclosed sequences.

In one aspect, the invention provides a recombinant DNA construct encoding a virus or viral vector. The vector according to the invention includes but not limited to lentivirus, retrovirus, adeno virus, adeno associated virus, hepatitis virus, herpes simplex virus, human papilloma virus and any other vector including but not limited to transposon-based vectors or the TALENs/ CRISPR-Cas9 based vectors that is known to a person skilled in the art.

In another aspect the present invention provides genetically modified population of immune cells isolated from a patient suffering from a disease, or a disorder transformed with a polynucleotide encoding CAR targeting CD20 wherein the said patient is suffering from a disease or disorder selected from a group consisting of cancer involving B cells, Acute Lymphoblastic Leukemia (ALL) or B cell Non-Hodgkin's Lymphoma (B-NHL), autoimmune disease, non-autoimmune inflammatory disease, transplantation and B cell-related disorder. In a preferred embodiment genetically modified population of immune cells are T lymphocytes or Natural Killer cells wherein the T lymphocytes are CD3+ (aß or ?d T cells) and a combination thereof.
The present invention further provides a process for preparing immune cells expressing the CAR, preferably anti-CD20 CAR of the invention, the steps of which include:

a) isolating immune cells from the patient or a suitable donor requiring treatment.
b) introducing the nucleic acid encoding the CAR into the immune cells; and
c) culturing the immune cells for expression of the CAR.

Process for preparing the CAR-T cells in accordance with the present invention
A typical process for autologous CAR-T cell manufacturing includes the following unit operations:
- isolation of T cells from an apheresis product;
- in vitro activation of T cells;
- genetic modification of T cells using viral or non-viral gene delivery methods;
- in vitro expansion of the CAR-T cells;
- harvesting and formulation for dose preparation.

The process development for CAR-T cells typically involves a permutation and combination of various steps as provided below:

- T cells were isolated from an apheresis product using a magnetic activated cell sorting technique or using a cell sorter in a close loop and aseptic manner (this could be negative selection or positive selection using beads or matrices with antibodies directed against CD3+ or CD4+ and CD8+ on T-cells).
- The isolated T cells were activated using anti-CD3 and anti-CD28 carrying beads or matrices
- Genetic modification of T-cells (to generate CAR-T cells) was achieved using an optimized viral or non-viral method that ensures integration of the CAR construct in the host cell genome.
- The CAR-T cells were expanded in culture with a unique combination of media, cytokines, growth factors, amino acid supplements and environmental parameters to get the desired cell number for dosing the patient.
- On achieving the requisite number of cells for patient dosing, the cells were washed and formulated with cryo-protectants, cryopreserved using a controlled rate freezing technique and stored below -150 °C. Such cryopreserved cells are ready for infusion into the same patient.

The process development at different stages involves assessment of the identity, quality and quantity of the CAR-T cells using flow cytometry, ELISA and/or microscopy-based assays. The resultant T-cell composition is ascertained using flow cytometry for different characteristics including immune memory, stem memory, effector memory, and regulatory phenotypes.

The foregoing examples are for better understanding of the invention and are illustrative of the invention and are strictly not construed to be limiting the scope of the invention

Example 1
Design and synthesis of plasmid DNA constructs for CAR expression in T cells:
in vitro transcribed (IVT) mRNA was used as a starting material to express CARs in T cells. This process leads to a transient expression of the CARs on the cells that lasts for ~96 hrs (~4 days). IVT was preferred over lentiviral vector-based transductions as it consumes lesser time. While using this method, all the assays must be completed within 4 days of transfection.
The design of the CAR constructs tested herein are provided in Table 1
Table 1: CAR Construct Design
S.no. Name CAR construct
1 IMN-001 CD20 LH – CD8a TM – 4-1BB – CD3?
2 IMN-004 CD20 HL – CD8a TM – 4-1BB – CD3?
3 IMN-017 Positive control CAR (CD19 CAR – IRES – GFP)
4 IMN-018 Positive control CAR (CD19 CAR)
5 IMN-019 Negative control (eGFP mRNA)

Amino acid sequences of the aforesaid constructs are provided below:
1. IMN-001: CD20 LH – CD8a TM – 4-1BB – CD3? (SEQ ID. 20):
MGWSLILLFLVAVATRVLSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

2. IMN-004: CD20 HL – CD8a TM – 4-1BB – CD3? (SEQ ID. 22):
MGWSLILLFLVAVATRVLSQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSGGGGSGGGGSGGGGSQIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

3. IMN-017: +ve control CAR (CD19 CAR – IRES – GFP) (SEQ ID. 27):
MGWSLILLFLVAVATRVLSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPSCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSRPSPSPPPNVTGRSRLEGRCAFVYMLFSTILPSFGNVRARKPGPVFLTSIPRGLSPLAKGMQGLLNVVKEAVPLEASRQTTSVATLCRQRNPPPGDRCLCGQKPRVDTPAKAAQPQCHVVSWIVVERVKWLTSSVFNKGLKDAQKVPHCMGSDLGPRCTCFTCVSRLKNVAPRTTGTWFSFEKHDDNMAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEHAIASGSALP
The corresponding nucleotide sequence is SEQ ID 28.

4. IMN-018: positive control CAR (CD19 CAR) (SEQ ID. 29):
MGWSLILLFLVAVATRVLSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSESKYGPPCPSCPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
The corresponding nucleotide sequence is SEQ ID 30.

5. IMN-019: GFP (SEQ ID. 31)
MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAINLCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAVCICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKGDVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEHAIASGSALP
The corresponding nucleotide sequence is SEQ ID 32.

The plasmid map for m-RNA expression by IVT is provided in Figure 1.

Method of cloning of CAR genes in plasmid for IVT:
PCR amplification of the pMRNAxp plasmid backbone:
PCR amplification of the pMRNAxp plasmid backbone was carried out as mentioned in Table 2 below. The oligonucleotide primers used for the PCR amplification are mentioned in Table 3 below. Six identical reactions were set-up for the amplification of the pMRNAxp vector backbone. PCR amplified pMRNAxp vector backbone was separated on 0.8% agarose gel and purified using Qiagen gel extraction kit according to the manufacturer’s instructions.

Table 2: PCR reaction details:

Table 3: Primer sequences:

The results for two reactions out of six are shown in Figure 2 of the present invention where Lanes 1-2 shows the PCR amplified pMRNAxp vector backbone (expected size: ~2.5 kb); Lane M indicates 1kb DNA Ladder (Thermo) (0.25, 0.5, 074. 1.9, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, 10.0kb?)

PCR amplification of Chimeric Antigen Receptor (CAR) gene inserts:
PCR amplification of the Chimeric Antigen Receptor (CAR-T) gene inserts was carried out as mentioned in Table 4 below.

Table 4: PCR reaction details

The oligonucleotide primers used for the PCR amplification is mentioned in Table 5 below. For IMN-001 and IMN-004, GeneArt supplied plasmids were used as the PCR template, while for IMN-017 and IMN-018, the in-house AH-CD19-CART/GFP plasmids were used as the PCR template.

Table 5: Primer sequences

PCR amplified Chimeric Antigen Receptor (CAR-T) gene inserts were separated on 0.8% agarose gel and purified using Qiagen gel extraction kit according to the manufacturer’s instructions. The results of the PCR amplification of the respective Chimeric Antigen Receptor (CAR) gene inserts are shown in Figure 3 and 4 where the Figure 3 shows PCR amplified CAR inserts from IMN-001 (lane 1) and IMN-004 (lane 2); M: 1Kb DNA marker. Figure 4 shows PCR amplified IMN-017 (lane 17) and IMN-018 lane 18); M: 1Kb DNA marker.

Infusion HD cloning of PCR amplified gene inserts into pMRNAxp plasmid backbone:
The Infusion HD cloning reaction for sub-cloning of PCR amplified gene inserts with pMRNAxp plasmid backbone was carried out as mentioned in Table 6 below.

Table 6: Reaction details
Components IMN-001 IMN-004 IMN-017 IMN-018
pMRNAxp (150ng) 1.1ul 3.2ul 5.0ul 3.0ul
CAR insert (180ng) 0.5ul 0.5ul 0.9ul 1.6ul
5X Infusion mix 2.0ul 2.0ul 2.0ul 2.0ul
Nuclease free water 4.4ul 4.3ul 2.1ul 3.4ul
Total 10ul 10ul 10ul 10ul
Incubate at 50°C for 30 min

Transformation and clone confirmation:
• 2.5 µL of the above infusion reaction mixtures was transformed into commercial E. coli DH5a chemically competent cells using the standard heat shock method.
• The cells were incubated on ice for 30min and transformation was carried out by heat shock method at 42°C for 90 sec.
• The transformed cells were further incubated on ice for 5 min and 1 mL of LB broth was added to the cell suspension.
• The transformed cells were regenerated for 1h at 37°C and 180 rpm.
• Post regeneration, the cells were centrifuged at 5000 rpm for 2 min.
• The cells were plated onto LB agar media containing Carbenicillin (100 µg/mL).
• Colonies were randomly selected, and sequence confirmed by Sanger sequencing.
• Plasmid isolation was then carried out from the sequence confirmed clones.
• The recombinant plasmids were confirmed by bi-directional Sanger sequencing.
Plasmid scaleup
• The sequence confirmed plasmids were re-transformed into commercial E. coli DH5a chemically competent cells as mentioned above.
• A single isolated colony was inoculated into 10 mL LB broth containing Carbenicillin (100 µg/mL) and incubated for 5-6 h at 37°C and 180 rpm as a pre-culture.
• The pre-culture was then inoculated at 1:100 dilution into 100 mL of Circle grow media containing Carbenicillin (100 µg/mL) and incubated overnight at 37°C and 180 rpm.
• Plasmid DNA was scaled-up using Endotoxin Free Quanta Maxi kit (Mdi) according to the manufacturer’s instructions.
• The scaled-up recombinant plasmids were sequence confirmed using bi-directional Sanger sequencing.

Details of the scaled-up plasmids mentioned in table 7 below.
Table 7: Plasmid Scale up details
Name Construct size (kb) Conc. (ng/ul) A260/280 Yield (ug)
IMN-001 4.0 888 1.8 355
IMN-004 4.0 1178 1.8 471
IMN-017 5.3 2237 1.8 894
IMN-018 4.0 753 1.8 301

IVT mRNA synthesis
The sequence confirmed recombinant plasmids were linearized using NotI restriction enzyme for generation of the IVT mRNA synthesis and IVT synthesis and mRNA synthesis and co-translational capping using Anti Reverse CAP Analog (ARCA) were performed.

This in vitro synthesized mRNA expressing the CARs becomes the raw material to generate the CAR-T cells. The data in Table. 7 proves the appropriate quality and quantity of the mRNA to generate CAR-T cells.

Example 2: Immunophenotyping of donor and mRNA transfections of the CAR constructs into T cells
5 healthy donors were screened for selection of 1 donor for all the assays. Donor was selected on the basis of phenotyping (Figure 5) and spontaneous cytotoxicity profile (Figure 6). Donor no. NHHV-17166 was chosen for further studies.

Immune phenotyping of T cell activation markers (CD25, CD69, CD71) from donor NHHV: 17166 was performed using flow cytometry. Donor profiling revealed that there was no activation marker expression in CD4 and CD8 cells, confirming no recent exposure or activation.

mRNA Transfection: Frozen PBMC’s were thawed and washed once with DPBS. The cells were then resuspended to a cell density of 50x106/mL with Easysep buffer and 50µl of T cell isolation cocktail was added to 50x106 cells, incubated for 5mins. It was followed by the addition of 50µl T cell isolation beads and incubated for 5 mins. The volume was made up to 2.5mL with Easysep buffer and incubated for 5 minutes in Stem cell magnet. After incubation, the supernatant was collected in a falcon and centrifuged to collect the cells. After T cell isolation, its purity was checked in Novocyte by gating on the CD3+, CD4+ and CD8+ cells against the PBMC’s used for isolation.

The cells were counted and resuspended in complete RPMI media containing CD3/CD28 Dynabeads (25µl /1 x106 cells) and IL-2. The cells were incubated for 72 h at 37 ?C in CO2 incubator. After incubation, the dynabeads were removed using the stem cell magnet and the activated T cells were harvested, resuspended in T buffer (provided in the Neon electroporation kit). T cells were transfected with mRNA constructs at a concentration of 10µg per million T cells and transfection was performed using the Neon electroporation system. The transfected cells were added to 6 well plates in complete RPMI media (without antibiotics). Un-transfected cells were kept for the control group.

Assessment of T cell purity post isolation and before transfection are provided in Figure 7.

Figure 7 shows the purity of the T cells isolated from the healthy donor NHHV-17166. Further it shows that the T cells were not pre-activated due to a prior infection and were activated only after the process of in vitro activation done as a part of the protocol.

Example 3: Protein L staining to assess CAR expression on T cells
T cells were isolated from corresponding donor PBMCs and activated with CD3/CD28 beads. After 72h, cells were transfected with the respective test and positive control constructs by Neon electroporation. Transfected cells were rested overnight. Transfection efficiency was determined by Protein L staining. Un-transfected cells were used as control.

Figure 8 and 9 provides Protein L staining for donor NHHV-17166 (untransfected) and Protein L staining after CD20 CAR mRNA transfections respectively.

Protein L staining as observed in Fig 8 and 9 shows the expression of the CAR on the cell surface. The antibody binds to the VL region of the murine scFv thus identifying the CAR on the cell surface. The staining is observed on CD3+, CD4+ and CD8+ cells for all the CARs expressed on the T cells.

Example 4: Target cell profiling
The target cells for cytotoxicity assays (cell lines Daudi, Raji and K562) were assessed for expression of the target CD20 and a surrogate B cell marker CD19. Daudi and Raji are B cell lines expressing CD19 and CD20, while K562 is a B cell line that does not express CD19 and CD20, hence is used as a negative control.

Figure 10 shows the characterization of the cell lines used in various assays. The said CAR targets CD20 expressed on B cells. Daudi and Raji are B cell lines that express CD20 as seen in the FLOW cytometry plots and histograms when stained with a CD20 recognizing monoclonal antibody. K562 is used as a negative control; this B cell line does not express CD20 as seen in this figure.

Expression of CD19 and CD20 are assessed as provided below Table 8
Table 8: Expression profile of cells.

A fixed number of Daudi, Raji and K562 cell lines were stained with CD19 and CD20 recognizing monoclonal antibodies. Plotting a standard curve using a limited dilution method, the receptor density on these cells was counted and classifies as ‘high expression’ when compared to K562 cells that do not express these receptors.

Example 5: Cytotoxicity Assay
Cytotoxicity assays were performed in DAUDI, RAJI and K562 cells at 48h timepoints. Effector cells were calculated based on transfection efficiency obtained from protein L staining. Cytokine analysis was performed on supernatants from 1:1 E:T ratios from 24h culture.

Target tumor cells (DAUDI, RAJI and K562) were harvested, washed and resuspended in DPBS. Cells were labelled with 1uM CTV and incubated for 15 minutes at 37°C. Cells were added with 5X volume of complete media and incubated for 5 minutes. Cells were centrifuged at 1500rpm for 5 minutes, counted and resuspended to the required cell density for assay. CTV labelling efficiency was determined by flow cytometry (Novocyte).

Effector cells were prepared based on the percent transfection efficiency for the respective constructs and resuspended in complete RPMI media. Cells were added to the plates at increasing effector ratios in 100µl volume. Labelled Target cells were added to them at 10,000 cells per well in 100µL volume. Appropriate control wells were included (Target cells alone +/- CTV).

Separate plates were seeded for 24h and 48h timepoints and incubated in CO2 incubator. At the end of 24h and 48h, plates were centrifuged, supernatants were collected and stored at -80°C until analysis. Cells were washed once in FACS staining buffer (HBSS + 5 % FBS) and resuspended in 7-AAD solution and proceeded for acquisition in Novocyte flow cytometer.

Percent target specific lysis was determined by gating on CTV+7AAD+ cells.

Figure 11 provides Cytotoxicity response of CD20 CART cells performed in DAUDI, RAJI and K562 cells at 48h timepoints.

The cytotoxicity and cytokine profiles from the co-culture assay (CAR-T + B cell lines) show a better cytotoxicity of the said CARs (IMN-001 and IMN-004) as compared to the CAR generated from CD20V mAb (CD20V-LH) (having nucleotide sequence of SEQ ID 33 and amino acid sequence of SEQ ID 34).

Example 6: Cytokine analysis:
For cytokine analysis, the supernatants stored from the cytotoxicity assay plates were used.
Supernatants from 1:1 E:T ratio wells were analyzed for cytokine levels by Multiplex kit from R & D systems for a panel of 14 cytokines. Assay was set up as per manufacturer’s instructions. Briefly, supernatants were diluted two-fold in incomplete RPMI media and incubated with Antibody coated magnetic microparticle cocktail for 2h at RT. Plate was washed 3x in wash buffer using a hand-held magnet and beads were incubated with detection antibody cocktail for 1h at RT. Plate was washed 3x in wash buffer and incubated with streptavidin-PE for 30 minutes at RT. Plate was washed and re suspended in 100µl wash buffer and read in MAGPIX instrument.

Cytokine levels were assessed by plotting a standard graph for all analytes using a 5-PL equation and expressed as pg/ml.

Levels of all cytokine analysis for DAUDI, RAJI and K562 cells are provided in Figure 14.

An array of cytokines were analyzed from the co-culture assays (CAR-T + B cell lines) and it is observed that the said/ claimed constructs consistently show higher levels of effector cytokines as compared to CAR derived from the CD20V mAb (CD20V-LH) (having nucleotide sequence of SEQ ID 33 and amino acid sequence of SEQ ID 34).
Figures 12A-12B and 13A – 13B show that IMN-001 and IMN-004 show a better effector cytokine profile and a lesser inflammatory cytokine profile making it more efficacious and lesser toxic as a potential therapy.

Example 7: Proliferation assay
For proliferation assays, the respective transfected effector cells were labelled with 1µM CTV. CTV labelling efficiency was determined by flow cytometry (Novocyte). Target cells (DAUDI, RAJI and K562) were re suspended in complete RPMI media and plated at 10,000 cells per well.

Labelled effector cells were seeded to the target cells to achieve 1:1 and 0.5: 1 E: T ratios. Effector cell numbers were calculated based on percent transfection efficiency. Appropriate control wells (effector cells alone, effector cells + CTV and effector cells - CTV) were included in the assay.

Plates were incubated for 72h in a 37°C CO2 incubator. At the end of 72h, plates were centrifuged and washed with FACS staining buffer. Cells were surface stained for CD3, CD4 and CD8 and incubated for 30 minutes in ice. At the end of 30 minutes, cells were washed 2X in FACS staining buffer (PBS containing 5% FBS) and re suspended in 7AAD solution and proceeded for acquisition in Novocyte.

Percent proliferation was calculated by gating on CD3+, CD4+and CD8 + T cells and CTV dilution was looked at all the three populations.

Figure 15A -15C shows that CAR-T cells proliferate on exposure to the specific CD20 antigen expressed on Daudi and Raji cells and do not proliferate in absence of the antigen as seen in the K562 cells that do not express CD20. This shows antigen specific cell proliferation of the CAR-T cells.
,CLAIMS:
1. A polynucleotide encoding a chimeric antigen receptor (CAR) construct comprising:
a) a signal peptide
b) an antigen binding domain;
c) a hinge region;
d) a transmembrane domain;
e) one or more co-stimulatory domain;
f) a signaling domain,
said antigen binding domain is selected from a CD20 binding domain (scFv) or an anti - CD20 binding domain (scFv);
said scFv comprising a heavy and a light chain in a format of heavy-light, light-heavy or heavy-heavy chains;
said signal peptide having nucleotide sequence of SEQ ID 24.

2. The polynucleotide encoding the chimeric antigen receptor (CAR) construct as claimed in claim 1, wherein said CAR provides high expression of IL-2 and low expression of IFN-?.

3. The polynucleotide encoding the chimeric antigen receptor (CAR) construct as claimed in claim 2, wherein release of said IL-2 release is about 5 to 10 times that of said IFN-?.

4. The polynucleotide encoding the chimeric antigen receptor (CAR) construct as claimed in any of the preceding claims, wherein said scFv is selected from (i) scFv comprising murine heavy chain nucleotide sequence of SEQ ID NO. 1 and murine light chain nucleotide sequence represented by nucleotide sequence of SEQ ID NO. 3; (ii) scFv comprising a heavy chain nucleotide sequence of human/mouse chimera having nucleotide sequence of SEQ ID NO. 5 and a light chain nucleotide sequence of human/mouse chimera having nucleotide sequence of SEQ ID NO. 7.
5. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein the hinge and transmembrane regions are selected from the group consisting of CD2, CD3, CD4, CD8, CD28 and CD45R.

6. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein one or more co-stimulatory domain is selected from the group consisting of CD27, CD28, 4-1BB, ICOS, OX40, CD27, CD30, CD40, CD40L, PD-1, KIR-DAP12 and TLRs.

7. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein the co-stimulatory domains are derived from CD28, 4-1BB or a combination of CD28 and 4-1BB.

8. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein the signaling domain comprises cytoplasmic domain of CD3 zeta.

9. The polynucleotide as claimed in any of the preceding claims, wherein said polynucleotide is an mRNA.

10. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein said polynucleotide encodes amino acid sequence of SEQ ID NO: 20 or 22.

11. The polynucleotide encoding the CAR construct as claimed in any of the preceding claims, wherein said polynucleotide comprises nucleotide sequence of SEQ ID NO: 19 or 21.

12. A polypeptide encoded by the polynucleotide as claimed in any of the preceding claims.

13. A genetically modified population of T cells transformed with a polynucleotide as claimed in any of claims 1 to 11, wherein the expression of said polynucleotide endows said population of T cells with antibody specificity.