WO2023161961A1

WO2023161961A1 - A system and method for t cell activation and immunomodulation

Info

Publication number: WO2023161961A1
Application number: PCT/IN2023/050176
Authority: WO
Inventors: Nilesh Kumar Joitram PATEL; Nency Mukeshbhai Bhimani
Original assignee: Lymphon Biologics Pvt. Ltd.
Priority date: 2022-02-24
Filing date: 2023-02-24
Publication date: 2023-08-31

Abstract

The present invention relates to a system and method for T cell activation and immunomodulation. Plasma chemical vapour deposition (PCVD) technique is used for the grafting of peptide- Histocompatibility Molecules (pMHCs) and co- stimulatory / co-regulatory molecules on the polymeric surfaces (E) of plates. This system and method is reproducible because it takes less time in production and is fully robotic so the human error is minimized and the results are reproducible. It is also robust, scalable and less time consumingmethod for grafting molecules. It provides a fast track approach to activate T cells and immunomodulation for treating various types of diseases like cancer, autoimmune disorder, infectious diseases, allergies, inflammatory condition.

Description

A SYSTEM AND METHOD FOR T CELL ACTIVATION AND IMMUNOMODULATION

FIELD OF THE INVENTION

The present invention relates to a system and method for T cell activation and immunomodulation. More specifically, it relates to a system and method for ex-vivo activation of antigen specific T cells and immunomodulation by cellular activation using pMHC and co-stimulatory and/or co-regulatory molecule/s immobilised on solid surfaces.

BACKGROUND OF THE INVENTION

Immunotherapy involving the priming and expansion of T lymphocytes (T cells) holds promise for the treatment of cancer, auto immune diseases and infectious diseases as well as transplant rejection in humans. Current studies of adoptive transfer in patients with viral infections and/or cancer involve the infusion of T cells that have been stimulated, cloned and expanded for many weeks in vitro on autologous dendritic cells (DC), virally infected B cells, and/or allogenic feeder cells. However, since adoptive T cell immunotherapy clinical trials often require billions of cells, existing in vitro T-cell expansion protocols are often inadequate to meet the demands of such trials.

Furthermore, optimal engraftment requires use of functional, and not senescent, T-cells, at the time of re-infusion. For clinical applications, it is important to ensure that the T cells have the desired functionality, i.e., that they proliferate, perform effector functions and produce cytokines in a desirable manner. In the natural setting, T cell activation is initiated by the engagement of the T cell receptor/CD3 complex (TCR/CD3) by a peptide-antigen bound to a major histocompatibility complex (MHC) molecule on the surface of an antigen- presenting cell (APC). While this is the primary signal in T cell activation, other receptor-ligand interactions between APCs and T cells are also required for complete activation. For example, TCR stimulation in the absence of other molecular interactions can induce a state of anergy, such that these cells cannot respond to full activation signals upon re- stimulation. In the alternative, T cells may die by programmed cell death (apoptosis) when activated by TCR engagement alone.

Accordingly, optimal functionality may be conferred via use of a second signalling molecule, e.g., a membrane -bound protein or a secreted product of the APC. In the context of membrane -bound proteins, such secondary interactions are usually adhesive in nature, reinforcing the contact between the two cells. Other signalling molecules, such as transduction of additional activation signals from the APC to the T cell may also be involved. For example, CD28 is a surface, glycoprotein present on 80% of peripheral T cells in humans and is present on both resting and activated T cells. CD28 binds to B7-1 (CD80) or B7-2 (CD86) and is one of the most potent of the known co-stimulatory molecules. CD28 ligation on T cells in conjunction with TCR engagement induces the production of interleukin-2 (IL-2). Secreted IL-2 is an important factor for ex vivo T cell expansion.

Co- stimulation of T cells has been shown to affect multiple aspects of T cell activation. It lowers the concentration of anti-CD3 required to induce a proliferative response in culture. CD28 co-stimulation also markedly enhances the production of lymphokines by helper T cells through transcriptional and post- transcriptional regulation of gene expression and can activate the cytolytic potential of cytotoxic T cells. Inhibition of CD28 co-stimulation in vivo can block xenograft rejection, and allograft rejection is significantly delayed.

Antibodies against CD3 are a critical component in many polyclonal T cell stimulation protocols. It was first demonstrated by Dixon et al., that immobilized anti-CD3 could mediate human T cell activation and expansion in the absence of cognate antigen recognition by the T cell receptor. Anti-CD3 initiates the activation and proliferation signalling cascade by crosslinking the components of the T cell receptor complex on the surface of T cells; thus, their requirement for immobilization. It was subsequently shown by Baroja et al., that a second signal from either an immobilized or soluble anti-CD28 stimuli was required for full T cell activation in combination with immobilized anti-CD3. Additional costimulatory signals provided through adhesion ligands such as CD2, LFA-1 and other TNF family members such as CD137 (4-1BB) can provide additional proliferative or survival signals to the T cells.

More importantly, the aforementioned effectors for stimulatory/co- stimulatory simulation have been widely applied in the context of manipulation of T-cells in vitro. In this context, a combination of anti-CD3 monoclonal antibody (first signal) and anti-CD28 monoclonal antibody (second signal) is most commonly used to simulate the APCs. The signals provided by anti-CD3 and anti- CD28 monoclonal antibodies are best-delivered to T-cells when the antibodies are immobilized on a solid surface such as plastic plates or sepharose beads.

Commercial products for T cell activation using tissue culture plates coated with immobilized anti-CD3 antibodies are available from CORNING (BioCoat™ T cell activation plates, Cat #354725) and are widely prepared by researchers using standard methods known to those familiar with the art.

A variety of surfaces and reagents containing anti-CD3 and anti-CD28 monoclonal antibodies have been developed for obtaining and expanding T cells for various applications. For instance, Levine et al. (The Journal of Immunology, vol. 159, No. 12: pp. 5921-5930, 1997) disclose tosyl-activated paramagnetic beads with a 4.5 micron (pM) diameter containing anti-CD3 and anti-CD28 monoclonal antibodies, which can be utilized to stimulate and proliferate T-cells and induce them to produce pro-inflammatory cytokines. Such beads are commercially available from Thermo-Fisher Scientific, Inc. under the trade name DYNABEADS CD3/CD28 T-cell expansion. U.S. Patent No. 6,352,694 describes a method for T cell activation and expansion using anti-CD3 and anti-CD28 antibodies immobilized on a 4.5 um diameter magnetic particle.

In Patent no WO 2017161092, the invention in various aspects provides for magnetic enrichment and/or expansion of antigen- specific T cells, allowing for identification and characterization of antigen- specific T cells and their T cell receptors (TCRs) for therapeutic and/or diagnostic purposes, as well as providing for production of antigen- specific engineered T cells for therapy. Incubation of paramagnetic nano-aAPCs in the presence of a magnetic field, either during enrichment and/or expansion steps, activates T cells through magnetic clustering of paramagnetic particles on the T cell surface.

As observed in abovementioned prior arts, the use of paramagnetic beads with immobilized monoclonal antibodies for expansion of T-cells in cell therapy requires separation and removal of the beads from the T-cells prior to patient infusion. This is a very labour-intensive process and results in cell loss, cell damage, increased risk of contamination and increased cost of processing. Because of the tight association of the immobilized monoclonal antibodies on the beads with the corresponding ligands on the surface of the target T-cells, the removal of the beads from the T-cells is difficult. The bead-cell conjugates are often separated by waiting until the T-cells internalize the target antigens and then using mechanical disruption techniques to separate the beads from the T-cells. This technique can cause damage to the T-cells and can also cause the ligated antigens on the T-cells to be removed from the cell surface. In addition, since activated T-cells are often most-desired for use in cell therapy protocols and the desirable properties of the cells are lost during the 24-72 hour waiting time, paramagnetic separation has a limited use in the adoptive cell-therapy setting.

Techniques for separation and purification of cells attached to paramagnetic beads are also unusable in the clinical context. For instance, the process of removing the paramagnetic beads after separation from the T-cells requires the passing of the cell/bead solution over a magnet. This process, while greatly reducing the number of beads remaining with the T-cells, does not completely eliminate the beads. Implantation of compositions containing beads into patients can cause toxic effects. The bead removal process also reduces the number of T-cells available for therapy, as many T-cells remain associated with the paramagnetic beads, even after mechanical disassociation. Some cell loss also occurs with respect to the T-cells that are manipulated but otherwise not bound to the beads because these cells are washed away prior to the internalization and/or mechanical removal step(s).

Early approaches used autologous APCs purified from patients — a long, labour-intensive process that is complicated by patient genetics and medical history. Such approaches use ex vzvo-differentiated APCs (examples include like autologous monocyte-derived dendritic cells, moDCs) to facilitate the enrichment of reactive cell clones, and often after multiple stimulations. Alternatively, cellular artificial APCs (aAPCs), such as those derived from the K562 leukaemia cell line, are more reliable than moDCs and can be genetically engineered to express an array of T-cell-activating ligands. These include co- stimulatory ligands or non-specific stimuli, such as agonistic anti-CD3 antibodies, to facilitate polyclonal expansion. However, such cell lines are prone to genetic drift and provide limited control of the level of expression of TCRs and co- stimulatory ligands.

The advancement in achieving fastest growing technology which is used clinically without any of the material infused in the body except body’s own cells and industrially suitable scalability of the system remains partly unsolved/ unresolved issue.

There are major drawbacks of these approaches like side effects, increased therapy development time, lower safety index, lower scalability i.e. lower ready- to-scale index and higher costs for the treatment. These approaches have a major disadvantage that the therapy development time increases because it becomes difficult to find the particular antigen binding site for binding of particular antibody and the antigen for the recent and unknown developing diseases.lt is being critical to recognize and meet the special demand of the cell therapy or immunomodulation without any changes in cells and the immunomodulatory molecules signalling outside the cells without entering the body. The methods that are available require too much time for developing strategy.

WO1995/034814A1 discloses a method for treating the surface of polymeric material of an assay device to increase the sensitivity of diagnostic assays and screening assays. The method involves the treatment of the surface of the polymeric material with unseparated oxygen plasma to increase the binding capability of an analyte or analyte binding number to such surface. The treated polymeric material is utilized as a diagnostic assay device for determining the amount or presence of an analyte binding member in test sample.

WO 2007/106212 discloses a plasma system which combines an atmospheric pressure plasma device coupled to a vacuum deposition chamber in order to deposit a biomolecule on a surface. The idea of combing vacuum chambers and atmospheric pressure plasma jets into one system represents a complex engineering challenge. Furthermore, exposing a biomolecule to vacuum can result in molecular damage, denaturation, and loss of functionality.

WO 2005/110626 describes the use of a non thermal plasma device to convert a liquid aerosol containing an active agent and a reactive monomer into a dry coating which contains both a polymer (produced by polymerizing the reactive monomer) and the active agent which is physically entrapped in the polymer coating. Similarly, W02005/106477 describes an atmospheric pressure non thermal plasma process that involves the introduction of reactive monomers and active agents into the plasma to produce a polymerized coating of the reactive monomer which entraps the active agent.

The requirement to induce reactions within the polymer precursor without damaging the active agent limits the types of molecules that can undergo controlled polymerization in plasma without loss of functionality. Typically, this requires the reactive precursor to contain a vinyl or cyclic structure, which can be preferentially reacted in the plasma. If molecules do not possess such functional groups, then they can be polymerized via bond breakage and fragmentation in other areas of the molecule, which can give rise to chemical alterations and loss of functionality. Some researchers have attempted to work around this limitation by chemically altering the molecule, e.g., adding reactive chemical functionality to the molecule. However, the resultant coating may lose some activity of the active agent and/or produce unforeseen consequence in clinical settings requiring detailed safety studies before such modified materials could be safely used in humans. The requirement to chemically modify the molecule also can increase the overall complexity and cost of the process. Furthermore, these types of processes require the active agent to be dissolved in a solvent, which may limit the applicability of these techniques. For example, the molecule may be partially or complete insoluble, or may require the use of organic solvents which are known to undergo plasma polymerization reactions and may therefore co - polymerize alongside the molecule, resulting in a coating that contains additional unwanted materials. These materials may produce negative biological reactions. For example, many biomolecules are biologically active due to their unique shape or conformation, wherein thermal energy can cause denaturation to render them inactive. Many pharmaceutical products suffer from similar limitations and cannot be directly exposed to plasma due to a loss of activity caused by chemical and/or conformational changes. As a result, researchers often have avoided intentional exposure of biomolecules to plasma sources as the thermal, electrical, UV and other active species within the plasma can induce irreversible chemical and / or conformation changes that would destroy the biological/pharmaceutical activity of the molecule. Hence, to overcome the abovementioned problems, it is desperately needed to develop a system and method for T cell activation and immunomodulation which is not subjected to such challenges.

Thus it is essential to design a novel system and method that meets this requirement and complies with all stipulated regulatory trials within a shortest period of time instead of any of time extensive and costly treatment. The present invention focuses on scalable models in shortest period of time for known and unknown debilitating conditions like COVID-19 or so.

OBJECT OF THE INVENTION

The principal object of the present invention is to provide a system and method for T cell activation and immunomodulation.

Further object of the present invention is to provide a well designed fast- track approach for activating T cells and immunomodulation.

Further object of the present invention is to provide a system and method for T cell activation and immunomodulation, wherein bioinformatics tools are used which would help in superfast development of therapy against known or unknown antigens.

Another object of the present invention is to provide a system and method for T cell activation and immunomodulation which helps the researchers to easily identify the particular antigen binding site for binding of particular antibody and antigen for the recent and unknown developing diseases.

Yet another object of the present invention is to provide a system and method for T cell activation and immunomodulation wherein it provides a universal approach to develop vaccines, immunotherapy for cancer, and autoimmune disorder, infectious diseases, allergies, and inflammatory condition therapies.

Further object of the present invention is to provide a system and method for T cell activation and immunomodulation which is specific for disease peptides. Another object of the present invention is to provide a system and method for T cell activation and immunomodulation wherein, T-cell engineering is not required unlike CAR-T approach.

Yet another object of the present invention is to provide a system and method for T cell activation and immunomodulation which provides higher ready- to-scale index and is cost-effective.

One more object of present invention is to provide a system and method for T cell activation and immunomodulation, where functionalities of biomolecules remain same and there is no requirement of any further steps for drying because dry technique such as atmospheric plasma polymerization technique is used.

One more object of present invention is to provide a system and method for T cell activation and immunomodulation, which is reproducible because it takes less time in production and fully robotic system so that human error is minimized and results are reproducible.

One more object of present invention is to provide a system and method for T cell activation and immunomodulation, which is biodegradable, sterile, ready to use, disposable, easy to handle, with reduced labour cost and production time.

Further object of the present invention is to provide a system and method for T cell activation and immunomodulation which could be quickly developed and cater off the shelf product and will comply all stipulated regulatory trials within a shortest period of time.

Further object of the present invention is to provide superfast approach for T cell activation and immunomodulation by employing the latest technology for immobilisation of the activating agents and which is least leachable.

Further object of the present invention is to provide a system and method for T cell activation and immunomodulation which is robust, scalable, avoids incubation, and is therefore less time consuming technique for grafting molecules.

One more object of the present invention is to provide real time sampling for the measurement of the activated cells. One more object of the present invention is to provide a system and method for T cell activation and immunomodulation that is simple in construction and reduce mechanical and operational complexity and commercially scalable.

SUMMARY OF THE INVENTION

The present invention relates to a system and method for T cell activation and immunomodulation.More particularly, the present invention relates to a well designed fast-track approach for activating T cells and immunomodulation, wherein bioinformatics tools are used to design MHC libraries, to recognize antigen specific peptide and CDR region to direct specific binding of antigen specific peptide on the cleft of MHC molecule for superfast development of therapy against known or unknown antigens. The pMHC and co- stimulatory or co-regulatory molecules at desired concentration are immobilised by plasma chemical vapour deposition (PCVD) technique on the polymeric surfaces of the plates. Leukepherised cells are brought in contact with the plate containing the immobilized pMHC and co- stimulatory or co-regulatory molecules. Antigen specific T cells get activated when interact with immobilized biomolecules. The present invention contains overall process of development of the activated plates used in T cell activation and immunomodulation. Plurality of such plates are arranged in such a manner that there is space between two successive plates. The T cell activation approach of the present invention provides higher ready-to- scale index and is cost-effective, biodegradable, sterile, ready to use, disposable, easy to handle, with reduced labour cost and production time. Further, it provides a new dimension to advanced therapeutics and to develop vaccines, cancer immunotherapy and auto-immune disorder therapies.

BRIEF DESCRIPTION OF DRAWINGS

It should be noted that the figures are only intended to facilitate the description of the preferred embodiments. The figures do not illustrate every aspect of the described embodiments and do not limit the scope of the present disclosure.

Other features and advantages of the present invention will become apparent to those of ordinary skill in the art through consideration of the ensuing description, the accompanying drawings, and the appended claims.

FIG. 1 depicts the system and method for T cell activation and immunomodulation .

FIG. 2 depicts the flow diagram of the overall manufacturing method of the fully functionalized polymeric plate by using Plasma Chemical Vapour Deposition (PCVD) technique.

FIG. 3 depicts the loss of biomolecules during immobilization as measured using ELISA on wash eluent sample following the immobilization procedure.

FIG. 4 depicts the graftability of the immobilized biomolecules on plate determined by the ELISA method which shows the amount of biomolecules that are attached to the plate.

FIG. 5(a to e) depicts cytokine expression of T cells due to the activation via immobilized biomolecules.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining the present invention in detail, it is to be understood that the invention is not limited in its application to the details of the parts illustrated. The invention is capable of other embodiments, as described above and of being practiced or carried out in a variety of ways. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not to limitation. The invention may have various embodiments and they may be performed as described in the following pages of the complete specification.

It is to be understood that the singular forms "a", "an", and "the" include plural reference 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, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof.

The present invention includes a system and method for fast track commercial development of functionalised plates which are used to modulate and activate T cells ex-vivo.

Further, it is to be understood that the terms as used herein, the term “MHC” used in the description means Major Histocompatibility Complex” which possess a set of closely linked polymorphic genes that code for cell surface proteins essential for adaptive immune system. As used herein the term “APC” used in the description means Antigen Presenting Cells. Further, as used herein the term “TCR” means T cell Receptors. The term “CD” used in the description means Cluster of Differentiation. Further, the term “pMHC" means Peptide loaded Major Histocompatibility Complex. Further, the term “CDR” used herein means Complementary Determining Regions. Further, the term “biomolecule” used herein includes polypeptides of either antigen or MHC or co- stimulatory molecules or co-regulatory molecules, and is not limited to a specific polypeptide but depends on the therapy.

The present invention relates to a system and method, where polymeric surfaces are coated with suitable biological moieties for immune cell modulation by transient interaction of immune cells to surface coated biological entities, wherein the said polymeric surfaces are coated with different concentrations of pMHC molecules using bioinformatics tool, loaded with antigen specific peptide, whereby the pMHC molecules are coated in combination with different concentrations of other co- stimulatory molecules and with different concentrations of other immune regulatory signalling molecules.

In some variation of the system and method, at least one antigen presenting complex comprises an MHC class I peptide binding cleft. In some of these variation, at least one antigen presenting complex comprising at least two fusion proteins, wherein a first fusion protein comprises a first MHC class I a chain and a first immunoglobulin heavy chain and wherein a second fusion protein comprises a second MHC class I a chain and a second immunoglobulin heavy chain. The first and second immunoglobulin heavy chains associate to form the MHC class I molecular complex. The MHC class I molecular complex comprises a first MHC class I peptide binding cleft and a second MHC class I peptide binding cleft.

In some variations of the system and method, at least one antigen presenting complex comprises an MHC class II peptide binding cleft. In some of these variations, the antigen presenting complex is an MHC class II molecule. In some of these variations, the antigen presenting complex is an MHC class II molecular complex comprising at least four fusion proteins, wherein (a) two first fusion proteins comprise (i) an immunoglobulin heavy chain and (ii) an extracellular domain of an MHC class lip chain; and (b) two second fusion proteins comprise (i) an immunoglobulin light chain and (ii) an extracellular domain of an MHC class Ila chain, wherein the two first and the two second fusion proteins associate to form the MHC class II molecular complex, wherein the extracellular domain of the MHC class lip chain of each first fusion protein and the extracellular domain of the MHC class Ila chain of each second fusion protein form an MHC class II peptide binding cleft. In some of these variations, the immunoglobulin heavy chain comprises a variable region. In some variations of the system and method, an antigenic peptide is bound to at least one antigen binding cleft. In some of these variations, the antigenic peptide is selected from the group consisting of a peptide of a tumour- associated antigen, a peptide of an auto antigen, a peptide of an alloantigen, and a peptide of an infectious agent antigen.

In some variations of the system and method, at least one T cell affecting molecule is a T cell co-stimulatory molecule. The T cell co- stimulatory molecule can be selected from the group consisting of CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BBL, CD70, CD30L, OX-40L, B7h (B7RP-1), CD40, LIGHT, an antibody that specifically binds to but not limited to CD28, an antibody that specifically binds to HVEM, an antibody that specifically binds to CD40, an antibody that specifically binds to 0X40, and an antibody that specifically binds to 4- IBB antibody that specifically binds to CTLA-4, antibody that specifically binds to ICOS, antibody that specifically binds to ICAM, antibody that specifically binds to receptors on T cell surface.

In some variations of the system and method, at least one antigen presenting complex is a non-classical MHC-like molecule. In some of these variations, the non-classical MHC-like molecule is a CD1 family member. The non-classical MHC-like molecule can be selected from the group consisting of CDla, CDlb, CDlc, CDld, and CDle.

A system for T cell activation and immunomodulationinvolves a bioinformatics tool configured for identification and characterization of an antigen specific peptide/s (A) for targeted diseases and synthesis ofa specific library of MHCs (B) which provide stable docking and binding of the selected antigen specific peptides (A);means for preparation of a peptide MHC (pMHC) pool by conjugating the antigen specific peptide (A) on its target MHC from the MHC pool (B)or by pulsing the antigen specific peptides (A) into peripheral blood mononuclear cells (PBMC) in a concentration range between 1-50 pg/ml; means for purification of the prepared target pMHC pool (C); and an atmospheric plasma chemical vapor deposition technique (3) for coating the solution of pMHC (C) and a co-stimulatory or co- regulatory molecule/s (D) (in a concentration range between 1 -100 pg/ml) on a polymeric surface/s (E) of a plate/s with plasma.

FIG. 1 illustrates the method for T cell activation and immunomodulation, which involves the following steps: i) Antigen specific peptides (A) are identified for targeted diseases using a bioinformatics tool. ii) The antigen specific peptides (A) are synthesized after identification in step i), and characterized using analytical method, i.e. IR and NMR. iii) A specific library of MHCs (B) is simultaneously designed using the bioinformatics tool and synthesized to provide stable docking and binding of the selected antigen specific peptide/s (A). iv) A peptide MHC (pMHC) pool (C) is prepared after synthesis in steps ii) and iii), by allowing the antigen specific peptide (A) to conjugate (1) on its target MHC from the MHC pool (B) or by pulsing the antigen specific peptides (A) in a concentration range between 1-50 pg/ml into peripheral blood mononuclear cells (PBMC). v) The target pMHC pool prepared in step iv) is purified (2). vi) Co- stimulatory molecules or co-regulatory molecules (D) are added in the pMHC pool (C) purified in step v), in a concentration range between 1 -100 pg/ml. vii)The solution of pMHC and the co-stimulatory or co-regulatory molecules prepared in step vi) is coated with plasma by an atmospheric plasma chemical vapor deposition technique (3) at 5 - 60°C temperature and 0.1 - 10 bar pressure for 5-100 seconds and immobilized on the polymeric surfaces (E) of the plates, for faster and effective grafting of biomolecules. (illustrated in detail in FIG. 2) viii) Multilayer polymeric surfaces (E) coated with pMHCs and the costimulatory or co-regulatory molecules (D) are prepared. Plurality of plates are arranged in a manner such that there is space between two successive plates. ix) Mass production (4) of the plates prepared in step viii) is done.

Leukepherised cells are brought in contact with the plate containing immobilized biomolecules. Antigen specific T cells interact with these immobilized biomolecules and get activated.

The MHC receptor component includes at least a portion of a Major Histocompatibility Class 1 protein, Major Histocompatibility Class 2 protein and Major Histocompatibility Class 3 protein wherein at least one MHC receptor component is encoded with at least one gene product of HLA-A, HLA-B, HLA - C, HLA-DPA1, HLA-DPB1, HLA-DRA, HLA-DRB1, HLA-DQA1 and HLA- DQB1 gene.

Further, the target pMHC pool is purified by using preferably affinity chromatography and RP-HPLC (Reverse-phase High Performance Liquid Chromatography) purification method.

Moreover, in the process step (viii); the polymeric surfaces (E) of the plates are selected but not limited to polystyrene (PS), polyethylene terephthalate (PET), polyether ether ketone (PEK), cyclic olefin copolymer (COC), acrylic, glass, ceramic, polypropylene (PP), polycarbonate (PC), poly dimethylsiloxane (PDMS).

FIG. 2 depicts in detail the flow diagram of the overall manufacturing process of the fully functionalized polymeric plate by using Plasma Chemical Vapour Deposition (PCVD) technique. While the plates are moulded, these undergo the PCVD treatment (3) using Oxygen, Helium, Argon, Nitrogen, Air, Carbon Dioxide, Ammonium or a combination thereof, 1-25 W/cm electrode power, 10-50 kHz frequency, 1-10 slm suction flow, 1-10 slm dilution flow, and polystyrene, PET or other polymeric substrate. After treatment, these molecules are self assembled on plate. Multiple plates are assembled and then sealed by laser treatment. This entire operation is done under closed system so the chances of contamination would be eliminated.

The system and method according to the present invention allows for more specific activation of antigen specific T cells as well as immunomodulation via immobilized biomolecules. Immobilization of biomolecules by conventional methods has some limitations including not being scalable for commercial applications.

The atmospheric plasma technique permits immobilization of biomolecules in terms of the quantity and quality of biomolecules bind, orientation, stability, and non-leachability. Furthermore, this technique eliminates a few limiting factors such as time-consuming and complexity.

This technique is more convenient to the previous traditional methods because atmospheric pressure plasma polymerization technique consists of the low temperature as well as low atmospheric pressure so, the chances of degradation of biomolecules are less and immobilization of biomolecules is higher as compared to the other traditional methods.

The present invention also has other unique features like a) biomolecules are covalently bonded to the polymeric surfaces so the chances of the washing of biomolecules are less hence, more biomolecules retain on polymeric surfaces. Furthermore, higher activation and immunomodulation takes place b) biomolecules functionalities also remain same because of using dry technique in the invention c) no requirement of any further steps for drying because of dry technique such as atmospheric plasma polymerization technique is used d) higher molecular weight molecules can be easily immobilized on the plates without degradation.

Step 1: Preparation of plate by PCVD method.

A plate was introduced in the space of plasma machine; a mixed atmosphere being present in the chamber, voltage was applied to said electrodes for generating and maintaining plasma in the space of chamber. Before introducing polystyrene material in the plasma chamber, samples were cleaned by sonication in ethanol for 15 min and dried under a laminar flow hood. Atmospheric pressure plasma discharge was obtained between two horizontally placed parallel electrodes. Oxygen was used as the carrier gas and was controlled by a mass flow controller with setting at 8 seem. Followed by Acrylic acid was used as the precursor and added to the inert carrier gas in the form of aerosol. For the coating of the plate, pMHC solution (3 pg/ml) was prepared by adding 10 ml of PBS to a glass bottle and pipetting 300 pl of co- stimulatory molecules into it. The solution was mixed by pipetting up and down. Glass beads (2 mm, Merck) were added in to the glass bottle until they reached the same level as the liquid in order to increase the volume. An ice pack was taped to the bottle containing the solution to maintain the temperature of the solution at 4°C during deposition after which it was stored in the fridge. With the help of 3D printed nozzle, solutions are coated on to the plate; two clean injectors were wetted by running the "cleaning atomizers" program for 5 minutes, using a suction flow of 8 slm. Deionized (DI) water was used for the pMHC and co stimulatory injector (Biomol injector) and acrylic acid (AA) for the acrylic acid injector (AA injector).

The plates were then oxygen plasma etched using equipment and selects the “Gas in” program. The etching was completed using a large round nozzle, 0 slm suction gas, 0 slm dilution gas, 70 W, 8 slm oxygen and 2 passes of the head. Once the etching was completed, a bottle containing 30 ml AA was attached to the AA injector. The head movement remained as previously set. A linker layer of AA was applied using a large round nozzle, 0.200 mbar pressure of suction gas, dilution gas 1:8 seem, 100 W, 5 mins and 0.400 m bar and 3 passes of the head over each well.

The extraneous variables were identified: suction gas, dilution gas, electrode power, electrode frequency, gas used for plasma, concentration of pMHC and co- stimulatory molecule solution, nozzle shape, nozzle-substrate separation, chemical used as reactive precursor, linker monolayer, substrate, substrate pre-deposition etching treatment. These variables were all kept constant in this trial.

The glass bottle containing 10 ml pMHC and co- stimulatory + PBS solution previously prepared was attached to the biomol injector and the suction flow and dilution flow were set as per given below.

Several ratio of suction and dilution flow were employed. Ratio of suction flow (1-5): Dilution flows (1-5) were taken.

The electrode power was set to 70 W and the frequency was kept constant at 30 kHz, the oxygen plasma gas flow was set to 65 slm. For the only condition, only the biomol injector was used, and the AA injector was disabled in the biomol + AA condition, both injectors were activated. The nozzle was inserted 3 mm into the well to be treated. Exposure times of 5 - 60 seconds were used, increasing in increments of 5 seconds. After each deposition, the head was moved to remove the nozzle from the well and insert it into the next one. Each condition was repeated in three wells. Once all the wells were treated, the plate was removed, and the appropriate incubation procedure was followed. Three different incubation conditions were tested: Wet incubation, Dry incubation and No incubation. The following buffers were used in these experiments. A blocking buffer consisting of 25 ml PBS (Dulbecco's phosphate buffered saline, Hlmedia) and 0.50 g of BSA (Bovine serum albumin, Sigma- Aldrich) was created, shaken to make sure that everything has dissolved. A storage buffer comprising 50 ml of ultra-pure water (Thermo Fisher), 55 g of mannitol (D-mannitol > 98%, Sigma-Aldrich) and 1 g of sucrose (D(+)-sucrose 99.5 %, Sigma- Aldrich). A washing buffer comprising 150 ml PBS and 3 pl of Tween80 (Sigma-Aldrich). Care was taken due to the viscosity of Tween80. A PTA buffer comprising 150 ml PBS, 0.15 g of BSA and 3 pl of Tween80. An analytical balance was used to determine the mass of components.

Wet incubation was prepared by adding 300 pl of PBS buffer to each well using a multipipette and the plate was stored for 24 h at 4°C. The solutions of the wells are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. 300 pl of PBS and 1 % BSA blocking buffer were pipetted into each well. The plate was then left to incubate for 2 hours at room temperature (RT) after which the wells were washed by flicking the plate over a sink.

Dry incubation was prepared by storing the plate at 4°C for 24 h. 300 pl of PBS and 1 % BSA blocking buffer was pipetted into each well. The plate was then left to incubate for 2 hours at RT and the wells were emptied by flicking the plate over a sink.

No incubation condition was prepared by pipetting 300 pl of PBS and 1 % BSA blocking buffer into each well. The plate was left to incubate for 2 hours at RT. The wells were then emptied by flicking the plate over a sink.

After incubation, the wells were washed by pipetting 300 pl of PBS and 0.05 % Tween 80 solution into them and emptying them on sink. This process was repeated 4 times. 175 pL/well of D-mannitol (100 g/L) and sucrose (20 g/L) storage buffer was added to each well and the plates were incubated for 3 mins at RT. The wells were then emptied by tapping over a sink. The remaining drops are removed by patting the plate on a paper towel. Finally, the plates were wrapped in tin foil and stored at -20°C in a freezer.

The plates were removed from the freezer. The plates were washed 4 times by pipetting 300 pl PBS and 0.05 % Tween 80 into the wells, which were then emptied by tapping over sink. 300 pL/well of 3ug/ml and 5 ug/ml of pMHC and co-stimulatory molecules solution in PTA was added and the plates were left to incubate at RT for 1 hour. One of the triplicate wells was always left empty to act as a blank. Each well was washed 4 times with PBS + 0.05 % Tween 80 and then emptied by tapping over sink. 100 pL/well of detection antibody in PTA, was added to each well. The plates were left to incubate at RT for 2 h. Each well was washed 3 times with PBS and 0.05% Tween 80 and then emptied by tapping over sink. 100 ng/ml streptavidin-HRP (Streptavidin horse radish peroxidase conjugate, Thermo Fisher) in PTA solution were added to each well. The plates were then left at RT for 1 h. Each well was washed 3 times with PBS and 0.05% Tween 80 and then emptied by tapping over sink. 100 pL/well of Tetramethylbenzidine (TMB) (TMB Liquid Substrate System for ELISA, Sigma-Aldrich) was added immediately after substrate addition, the plate was inserted into the spectrophotometer and absorbance of light set at 370 nm and 652 nm wavelengths then measured every 5 minutes for 1 hour after TMB addition.

FIG. 3 illustrates the loss of biomolecules during immobilization(pg) as measured using ELISA on wash eluent sample following the immobilization procedure. After immobilization, pMHC and anti CD28 antibodies were incubated with buffer solution for 4 hours to determine if any significant amount of biomolecules leach off in to the buffer or not. No significant loss of biomolecules was observed over the time of course of experiment. As described in the graph, the amount of biomolecules that leach in the second wash is less than the first wash.

FIG. 4 illustrates the graftability of the immobilized biomolecules on the plate. This was determined by ELISA method to show the amount of biomolecules that are attached to the plate. This graph describes the amount of biomolecules actively immobilized through the plasma atmospheric method. The amount of pMHC and CD 28 biomolecules that are immobilized are determined by the ELISA technique. As described in the graph, different concentrations of biomolecules were immobilized and absorbance was taken with the help of ELISA reader. At the concentration of 5 ug/ml showed better result than any other concentration.

Step 2: Evaluation of the plate with patient blood.

For the evaluation of the coated plate, written consent was obtained from healthy donors. 10 mL of blood sample was drawn from two healthy donors in lithium heparinized tubes. These tubes were mixed well and stored at 4°C until the PBMCs isolation procedure was initiated. For Peripheral blood mononuclear cell (PBMC) isolation, The samples were under laid as a 1:1 volume with Ficoll-Paquemedium (HISEP LSM 1073) equal to the sample volume in sterile condition of the biosafety cabinet. There After, these sample centrifuged at 2500 rpm for 30 minutes at room temperature with the brake OFF. After centrifugation, PBMCs present in the buffy layer were harvested from Ficoll-Paque medium.Then the cells were washed with PBS and centrifuged at 2000 rpm for 5 minutes at 4°C for two times and the supernatant was discarded. The cell pellet was resuspended in 2 ml TexMACS™ medium, from that 10 ul cells were taken and diluted to 500 ul PBS and cell count analysis was performed on Miltneyi Biotec’s MACSQuant - flow cytometer. The cell count was calculated as per dilution factor = cell count x 50 x 2000 .

For T cell activation; Antigen presenting cells (APCs)were prepared in a ratio of 1:1 of activation beads and antibodies from Miltneyi Biotec’s T cell expansion kit and the experiment was carried out in 24 well culture plates. Cells were added in the well as per cell count and serum was added as 5% of cells. APCs were added in a ratio of 1:2 to that of the cells. TexMACS ™ medium was added making a final volume of 1 ml. Culture plates were incubated in a CO 2 incubator at room temperature for 15 hours. After Activation of T Cells, Analysis could be done using Flow cytometry. So, After incubation, samples were centrifuged at 2000 rpm for 5 minutes at 4°C.5O pl was taken from the supernatant for cytokine analysis and the remaining was discarded and the pellet was resuspended in 150 pl. The samples were divided into tubes and labelled with CD69 including control. In each tube, 1 pl of CD3 (Anti-human Reafinity PE -CAT. NO. - 130-113-139) was added. 1.5 pl of each CD69 (Anti-human Reafinity APC -CAT.NO. - 130-112-614)) antibodies were added as per labeled tubes. Then, samples were incubated at 4°C for 30 minutes and wrapped with aluminium foil and adjusted up to 200 ul with PBS and analyzed on flow cytometry at appropriate channels. When we performed cytokine analysis, Cytokine bead array was performed by using Miltenyi Biotec’s cytokine assay kit. In this, 50 pl sample was taken into a 1.5 ml polystyrene tube and resuspended 15 pl capture beads at least 30 s and vortexed before use for well mixing and incubated for 2 hr at room temperature in dark. After 2h incubation, 0.5 ml of MACSPlex Buffer was added to each tube and centrifuged at 3000xg for 5 minutes. The supernatant was carefully aspirated by leaving 20 ul in the tube. The MACSPlex Capture Bead pellet was resuspended in each tube by adding 0.5 mL of MACSPlex Buffer and pipetted up and down and centrifuged at 3000xg for 5 minutes. The supernatant was carefully aspirated by leaving 20 ul in the tube.15 ul of detection reagent was added to each tube and incubated for 1 h protected from light. 0.5 ml of MACSPlex buffer was added to each tube and centrifuged at 3000 x g for 5 minutes. The supernatant was aspirated carefully by leaving 20 ul in the tube. The pellet was resuspended in each tube with 200 pl of MACSPlex buffer. The samples were analyzed by Miltneyi Biotec’s MACs Quant flow cytometer at PE and FITC channels.

FIG. 5 (a to e) illustrates different cytokine expression measurement of T cell activation on the immobilized plate of biomolecules. Figure 5(a) describes the CD69 expression marker. CD69 is rapidly induced on the surface of T lymphocytes after TCR/CD3 engagement, activating cytokines and polyclonal, mitogenic stimulation. Transcriptional expression of the CD69 gene is detected early after activation (30-60 min). So, when the T lymphocytes from the leukapharesis were contacted with the immobilised biomolecules, CD69 molecules showed expression. As described in the graph, Control was taken in which no such types of activators were present. Control with standard CD3 and CD28 antibody which are marketed T cell activators and others are the different peptide samples. The expression of CD69 in peptide 2(P2) is comparatively higher than any other peptide and shows markedly result than the marketed CD3 and CD28 product. These peptides are disease specific peptides, so the result shows that the T cell activation is specific. Figures 5 (b, c, d, e) describe the amount of cytokine production while T cells were getting activated. In figure 5(b), GMCSF is the critical cytokine which is produced by the activated T cells and form colonies of CD4+. Concentration of GMCSF is notable in the peptide stimulated T cells; as mentioned in the graph, P2 peptide gave higher concentration of the GMCSF as compared to the other peptides.

Figure 5(c) shows that IL2 secretion which is the marker of the activation of T cells. Peptide 2 shows comparatively higher secretion than the other peptides.

Figure 5(d) shows IFN gamma secretion when CD4 T helper type 1 (Thl) cells and CD8 cytotoxic T cells are predominantly activated. P2 peptide shows higher secretion as compared to the other peptides.

Figure 5(e) ( i and ii) shows secretion of the granzymes and perforin which are important for cytotoxicity and CD8+ T cells secrete these cytotoxic molecules for killing cells. Amount of the granzymes and perforin higher when T cells attack the cancerous cells. As per the graph, the P2 peptide showed markedly higher secretion than other peptides. As compared to the other techniques, the system and method of present invention is reproducible because it takes less time in production and it is fully robotic so the human error is minimized and the results are reproducible. It is also robust, scalable and less time consuming for grafting molecules.

As described by many patents and published paper as well as marketed products, all products are introduced in to the body and they give adverse effects so addressing this challenge by not introducing any of the molecules into the body can be overcome by this invention.

This system and method hereof can readily be adapted or extended to alter the responses of cells and in a variety of different ways (for example, increasing or decreasing their responses to a variety of stimuli).

The invention has been described with reference to specific embodiment which is merely illustrative and not intended to limit the scope of the invention as defined in the present complete specification.

List of Reference Numerals:

1. Antigen specific peptide/s (A)

2. Syn-MHC pool (B)

3. Syn-pMHC pool (C)

4. Co- stimulatory or co-regulatory molecules (D)

5. Polymeric surface (E)

6. Conjugation of antigen peptide on MHC (1)

7. Purification of target pMHC (2)

8. Plasma coating on polymeric surface via PCVD technique (3)

9. Mass Production (4)

Claims

WE CLAIM:

1. A system for T cell activation and immunomodulationcharacterized by a bioinformatics tool configured for identification and characterization of a known or an unknown antigen specific peptide (A) for targeted diseases and synthesis of a specific library of MHCs (B) which provide stable docking and binding of the selected antigen specific peptides (A); means for preparation of a peptide MHC (pMHC) pool by conjugating the antigen specific peptide (A) on its target MHC from the MHC pool (B) or by pulsing the antigen specific peptides (A) into peripheral blood mononuclear cells (PBMC);means for purification to purify the prepared target pMHC pool (C); and an atmospheric plasma chemical vapor deposition technique (3) for coating the solution of pMHC (C) and co-stimulatory or co-regulatory molecules (D) on polymeric surfaces (E) of plates with plasma.

2. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the MHC receptor component includes at least a portion of a Major Histocompatibility Class 1 protein, Major Histocompatibility Class 2 protein and Major Histocompatibility Class 3 protein.

3. The system for T cell activation and immunomodulation as claimed in claim 1, wherein at least one MHC receptor component encodes at least one gene product of HLA-A, HLA-B, HLA -C, HLA-DPA1, HLA-DPB1, HLA-DRA, HLA-DRB1, HLA-DQA1 and HLA-DQB1 gene.

4. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the means for purification of the target pMHC pool is preferably affinity chromatography and RP-HPLC.

5. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the antigen specific peptide (A) is selected from the group consisting of a peptide of a tumour-associated antigen, a peptide of an auto antigen, a peptide of an alloantigen, and a peptide of an infectious agent antigen.

6. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the antigen specific peptides (A) are pulsed in a concentration range between 1-50 pg/ml into the peripheral blood mononuclear cells (PBMC).

7. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the co-stimulatory or the co-regulatory molecules (D) is selected from the group consisting of CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BBL, , CD70, CD30L, OX-40L, B7h (B7RP-1), CD40, LIGHT, antibody that specifically binds to but not limited to CD28, antibody that specifically binds to HVEM, antibody that specifically binds to CD40, antibody that specifically binds to 0X40, and antibody that specifically binds to 4-1BB antibody that specifically binds to CTLA-4, antibody that specifically binds to ICOS, antibody that specifically binds to ICAM, antibody that specifically binds to receptors on T cell surface.

8. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the co-stimulatory molecules or co-regulatory molecules (D) are added in a concentration range between 1-100 pg/ml.

9. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the solution of pMHC (C) and the co-stimulatory or the co- regulatory molecules (D) is coated and immobilized on the polymeric surfaces (E) of the plates with plasma by the atmospheric plasma chemical vapor deposition technique (3) at 5- 60°C temperature and 0.1 - 10 bar pressure for 5-100 seconds. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the plates undergo the PCVD treatment (3) using Oxygen, Helium, Argon, Nitrogen, Air, Carbon Dioxide, Ammonium or a combination thereof, 1-25 W/cm² electrode power, 10-50 kHz frequency, 1-10 slm suction flow, 1- 10 slm dilution flow. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the polymeric surfaces (E) are coated with biological moi eties for immune cell modulation by transient interaction of immune cells to surface coated biological entities, with different concentrations of pMHC molecules using bioinformatics tool, loaded with specific antigen peptide, with different concentrations of other co-stimulatory molecules and with different concentrations of other immune regulatory signalling molecules. The system for T cell activation and immunomodulation as claimed in claim 1, wherein the polymeric surfaces (E) of the plates are selected from polystyrene (PS), polyethylene terephthalate (PET), polyether ether ketone (PEK), cyclic olefin copolymer (COC), acrylic, glass, ceramic, polypropylene (PP), polycarbonate (PC), polydimethylsiloxane (PDMS). A method for T cell activation and immunomodulation comprising the following steps: i) identifying a known or an unknown antigen specific peptide (A) for targeted diseases using a bioinformatics tool; ii) synthesizing the antigen specific peptides (A) after identification in step i),and characterizing them using an analytical process; iii) designing a specific library of MHCs (B) simultaneously using the bioinformatics tool and synthesizing to provide stable docking and binding of selected antigen specific peptides (A); iv) preparing a peptide MHC (pMHC) pool by allowing the antigen specific peptide (A) to conjugate (1) on its target MHC from the MHC pool (B) or by pulsing the antigen specific peptides (A) into peripheral blood mononuclear cells (PBMC); v) purifying the target pMHC pool (C) prepared in step iv); vi) adding co-stimulatory molecules or co-regulatory molecules (D) in the pMHC pool (C) purified in step v); vii) coating the solution of pMHC (C) and the co-stimulatory or the co- regulatory molecules (D) prepared in step vi) with a plasma by an atmospheric plasma chemical vapour deposition technique on polymeric surfaces (E) of plates. The method for T cell activation and immunomodulation as claimed in claim 13 step ii), wherein the analytical process is selected from, but not limited to ELISA, and Flow cytometry. The method for T cell activation and immunomodulation as claimed in claim 13 step ii), wherein the antigen specific peptide (A) is selected from the group consisting of a peptide of a tumour-associated antigen, a peptide of an auto antigen, a peptide of an alloantigen, and a peptide of an infectious agent antigen. The method for T cell activation and immunomodulation as claimed in claim 13 step iv), wherein theantigen specificpeptides (A) are pulsed in a concentration range between 1-50 pg/ml into the peripheral blood mononuclear cells (PBMC). The method for T cell activation and immunomodulation as claimed in claim 13 step vi), wherein the co-stimulatory or the co-regulatory molecules (D) is selected from the group consisting of CD80 (B7-1), CD86 (B7-2), B7-H3, 4- 1BBL, CD 70, CD30L, OX-40L, B7h (B7RP-1), CD40, LIGHT, antibody that specifically binds to but not limited CD28, antibody that specifically binds to HVEM, antibody that specifically binds to CD40, antibody that specifically binds to 0X40, and antibody that specifically binds to 4-1BB antibody that specifically binds to CTLA-4, antibody that specifically binds to ICOS, antibody that specifically binds to ICAM, antibody that specifically binds to receptors on T cell surface.

18. The method for T cell activation and immunomodulation as claimed in claim 13 step vi), wherein the co-stimulatory moleculesor co-regulatory molecules (D) are added in a concentration range between 1 -100 pg/ml.

19. The method for T cell activation and immunomodulation as claimed in claim 13 step vii), wherein the solution of pMHC (C) and the co-stimulatory or the co-regulatory molecules (D) is coated and immobilized on the polymeric surfaces (E) of the plates with plasma by the atmospheric plasma chemical vapor deposition technique (3) at 5 - 60°C temperature and 0.1 - 10 bar pressure for 5-100 seconds.

20. The method for T cell activation and immunomodulation as claimed in claim 13 step vii), wherein the plates undergo the PCVD treatment (3) using Oxygen, Helium, Argon, Nitrogen, Air, Carbon Dioxide, Ammonium or a combination thereof, 1-25 W/cm² electrode power, 10-50 kHz frequency, 1-10 slm suction flow, 1-10 slm dilution flow.

21. The method for T cell activation and immunomodulation as claimed in claim 13 step vii), wherein the polymeric surfaces (E) of the plates are selected from polystyrene (PS), polyethylene terephthalate (PET), polyether ether ketone (PEK), cyclic olefin copolymer (COC), acrylic, glass, ceramic, polypropylene (PP), polycarbonate (PC), polydimethylsiloxane (PDMS).