WO2016207911A4

WO2016207911A4 - System for cytoreduction of circulating cancer cells from blood and a method thereof

Info

Publication number: WO2016207911A4
Application number: PCT/IN2016/050194
Authority: WO
Inventors: Pankajkumar K. CHHATRALA; Sheetal PARIDA; Mahitosh Mandal
Original assignee: Chhatrala Pankajkumar K; Parida Sheetal; Mahitosh Mandal
Priority date: 2015-06-22
Filing date: 2016-06-22
Publication date: 2017-04-27
Also published as: WO2016207911A2; WO2016207911A3

Abstract

A system for selective separation of cancer cells from blood without losing normal cells comprising:an incubator unit for feeding in the blood for marking the cancer cells for separation involving nanoparticle; a separation unit for separating the nanoparticle marked cancer cells from normal cells and a recycling chamber for regenerating the nanoparticles for subsequent use for marking cancer cells. The method for carrying out cyto-reduction of cancer cells by is capable of continuous separation of cancer cells from blood for therapeutic purpose. Advantageously the advancement has potential to be used as adjuvant therapy with chemotherapy in leukemia patient

Claims

AMENDED CLAIMS received by the International Bureau on 02. March. 2017 (02.03.2017)

[Claim 1] A system for therapeutic application of selective separation of cancer cells from blood without losing normal cells comprising:

an incubator unit for feeding in the blood for marking the cancer cells for seperation involving nanoparticle;

a seperation unit for seperating the nanoparticle marked cancer cells from normal cells; and

a recycling chamber for regenerating the nanoparticles for subsequent use for marking cancer cells.

[Claim 2] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claiml wherein the nanoparticles are preferably paramagnetic nanoparticle coated with biocompatible polymer and functionalized by tagging specific antibody on polymer surface.

[Claim 3] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1 and 2, wherein the incubator comprises microchannel and static mixture inside micro-channels so that for mixingblood properly with anti-coagulant and nanoparticles whereby cancer cells are attached to nanoparticle on cell surface due to antigen antibody reaction.

[Claim 4] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1 to 3 wherein in the separation unit comprises

at least one blood channel for carrying the blood with said marked cancer cells and at least one spaced transfer fluid channel carrying a transfer fluid in opposite direction and having an adjacent magnetic field to attract paramagnetic nano-particles, said central blood carrying channel and said transfer fluid channel connected through plurality of small branches;

pressure controller for desired velocity of flow of blood in said blood channel and said transfer fluid channel such that there is no flow of blood from blood channel into fluid channel while the paramagnetic nanoparticles in blood stream get attracted by the magnetic field adjacent said fluid channel and thus get carried over and separated into said transfer fluid channel .

[Claim 5] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1 to 4 comprising 15 said separation unit comprises a central blood flow channel;

peripheral fluid flow channels with adjacent magnetand carrying transfer buffer in direction to said blood flow ;

said central blood channels connected to peripheral channels with small braches whereby cancer cells with paramegnatic nano particles attached are attracted by the said field and separated from said blood channel to the peripheral fluid channels for onward tranfer to said recycling chamber.

[Claim 6] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1-5 wherein the transfer fluid comprises

transfer buffer selected from plasma, serum or electrolyte having similar electrolytes and proteins same as blood and maintains osmotic and oncotic pressure ;

pressure and velocity of blood and transfer buffer in channels is maintained by said pressure controller in such a way so that no transfer of blood component takes places from central channel to peripheral channels resulting into blood-transfer fluid interface through which cancer cells can be passed.

[Claim 7] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1-6 wherein the recycling chamber include capillaries put into parallel connections and are covered by electro-magnet that switch on/off periodically to perform trapping, eluting preferably with eluting buffer and washing process of nanoparticles for its regeneration from the nanoparticle marked cancer cells and its subsequent use preferably involving valve based cooperative control to maintain uninterrupted flow of separating buffer through recycling chamber.

[Claim 8] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1-7 wherein the eluting buffer is either acidic or basic buffer selected from lOOmM Glycine, pH2.5 (acidic pH), 1M Triethanolamine; TEA (basic pH), 4M MgC12 (high salt), 1M NaCl/PBS (high salt) or any other eluting buffer that can be used for desired resut.

[Claim 9] A system for therapeutic application of selective separation of cancer cells from blood as claimed in claims 1-8 wherein said recycling chamber sequentially activate magnet ON, such that all paramagnetic nanoparticles attached with cancer cells are trapped in chamber to 16 allow transfer buffer to pass on for next cycle (Step-1), before going into next cycle, transfer buffer is filtered through micro porous filter to remove any cells or cellular remnants and also when magnet is still ON, same capillaries are flushed with eluting buffer so that cancer cells are detached from antibody and removed(Step-2), and finally when the magnet is switched OFF so that nanoparticles(set free in the step 2) are released from the wall, washed and sent again into incubation chamber involving preferably physiological buffer(Normal saline or Ringer lactate solution).

[Claim 10] A method of therapeutic application for carrying out cyto -reduction of cancer cells in blood comprising:

marking the cancer cells in blood with naoparticles;

seperating the nanoparticle marked cancer cells from normal cells under magnetic influence and providing the blood free of said cancer cells and restoring the blood with normal undamaged biologically functional cells;

regenerating the nanoparticles from the nanoparticle marked cancer cells.

[Claim 11] A method of therapeutic application for carrying out cyto -reduction of cancer cells as claimed in claim 10 comprising:

marking the cancer cells in blood with nanoparticles comprise the step : mixing blood mixes properly with anti-coagulant and nanoparticles in an incubator preferably including microchannel and static mixture inside micro-channels whereby cancer cells are attached to nanoparticle on cell surface due to antigen antibody reaction;

separation step wherein only the nanoparticle marked cancer cells are separated from blood under magnetic force; and

regeneration step to regenerate the nanoparticles from the nanoparticle marked cancer cells for subsequent use.

[Claim 12] A method of therapeutic application for marking the cancer cells in blood with nanoparticles comprise the step : mixing blood mixes properly with anti-coagulant and nanoparticles in an incubator preferably including microchannel and static mixture inside micro- channels whereby cancer cells are attached to nanoparticle on cell surface due to antigen antibody reaction.

[Claim 13] A method of therapeutic application for regeneration of nanoparticles from the nanoparticle marked cells present in a fluid/buffer medium involving a recycling chamber having number of capillaries put into 17 parallel connections and covered by electro-magnet that switch on/off periodically to perform trapping, eluting and washing process of nanoparticles including the steps of:

Step- 1 : when magnet is ON, all paramagnetic nanoparticles attached with cells are trapped in chamber allowing fluid/buffer to pass on for next cycle is filtered through microporous filter to remove any cells or cellular remnants;

Step-2: magnet is ON, same capillaries are flushed with eluting buffer, cells are detached from antibody and removed.

Step 3: when the magnet is switched OFF the nanoparticles set free in the step 2 are released from the wall, washed and sent again into incubation chamber using physiological buffer (Normal saline or Ringer lactate solution).

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STATEMENT UNDER ARTICLE 19

This is with reference to the International application PCT/IN2016/050194, ISR/Written Opinion (WO) date of January 03, 2017. The ISR/WO has mentioned that the present application lacks novelty and inventive step in view of US20120045828 (hereinafter 'Dl ') and the applicants respectfully disagree with the ISR/WO and humbly submit as below:

Claims have been thoroughly amended based on the originally filed claims and the descriptions as filed to make the claims more definitive. The amended claims are attached herewith.

The present invention resides in therapeutic application in leukacytopheresis by selective separation of cancer cells from blood in closed loop manner.

The main aspect of the present invention is therapeutic application in leukacytopheresis by selective separation of cancer cells from blood in closed loop manner, without the loss of the normal cells wherein the cancer cells are selectively marked with the paramagnetic nanoparticles coated with biocompatible polymer and functionalized by tagging specific antibody on the polymer surface which will bind specifically to the cancer cells and will be separated selectively from the normal cells and the undamaged biologically functional cells will return back to the patient.

Further Dl discloses an automated robotic device that isolates circulating tumor cells (CTCs). The isolation procedure which consists of a device for capture and isolation of target cells from limited quantity of sample from a mixed cell population wherein the target cells are labeled with magnetically responsive material to form labeled target cells. The cited invention is the field of cell isolation, including the isolation of cells from peripheral 19

circulation, labeling cells of interest magnetically, and using an automatable apparatus to immobilize and isolate viable labeled cells for further testing and culture.

The present invention however comprises a therapeutic application in leukacytopheresis system by selective separation of cancer cells from blood without losing normal cells comprising; an incubator unit for feeding in the blood for marking the cancer cells for separation involving nanoparticle; a separation unit for separating the nanoparticle marked cancer cells from normal cells; and a recycling chamber for regenerating the nanoparticles for subsequent use for marking cancer cells which also facilitates recycling of the substrate after each separation .

It is also to be noted that the cited prior art Dl is mainly for the isolation of circulating tumor cells (CTCs) and capturing of the targeted sample cells wherein the sample quantity is 9-10 ml which can only be used in diagnostic purpose. While the present invention is a continuous separation by leukacytopheresis system which can separate cancer cells from the normal cells enabling cleaning the blood and return undamaged biologically functional cells to the patients thus having significant therapeutic application.

Thus none of the cited art alone or in combination teaches the therapeutic application in leukacytopheresis by selective separation of cancer cells from blood in closed loop manner. Therefore, the present invention is both novel and inventive in view of Dl.

Amended claim annexed hereto may be considered as amended claims with Statement under Article 19 of the PCT.