Title: Reagents and Kits for Enrichment of Desired Cells Prior to Fluorescence Activated Cell Sorting

Inventor: Thomas Russell

Assignee/Applicant: Russell Biotech, Inc.

Cross Reference to Related Applications:

This application claims benefit of and priority to U.S. Provisional Patent Application No. 62/635,586, filed February 27, 2018, where permissible incorporated by reference in its entirety.

Background:

1. Field of the Invention: The invention provides improved nano/microparticle separation methods and kits for enriching a desired cell population prior to Fluorescence

Activated Cell Sorting (FACS) by removing undesired cell populations and yielding the desired cell population in very high yield prior to FACS. The invention relates more specifically to kits and reagents to improve FACS. 2. Description of related art: FACS provides a method to sort a desired cell population to a high degree of purity (Ref 1-5; 7). The purity of the final product is limited by the percent the desired cell population is in the starting sample. The lower the percentage the desired cell population is in the starting material the less pure the desired cell population following FACS. Also, the lower the percentage of the desired cell population in the starting sample the longer FACS takes. This is a significant issue as viability of the purified cell population can decrease the longer the sort times.

FACS involves labelling the desired cell population with a fluorescent label attached usually to a monoclonal antibody that binds to the surface of the desired cell population. The fluorescent cells are then sorted away from the non-fluorescent cells yielding the desired cells physically separated from the starting sample. The isolated cells can then be used in various biological experiments.

Magnetic separation technologies exist in the art that can be used as a pre-step prior to FACS (Ref 6). The particles are composed of metal oxides 50nm or less in diameter i.e. inorganic compounds (primarily iron oxides) imbedded in non-magnetic material as described in 5,411,863 (Miltenyi); 5,466,574 (Liberti); 4,654,267 (Ugelstad), 4,707,523 (Chang) and Reference 8. Magnetic particles available commercially are by and large made of these inorganic iron oxides. These magnetic particles of the art are less than desirable since they yield a desired cell population in unacceptable yield (Fig 1) prior to FACS analysis.

There is definitely a need for a pre-step procedure prior to FACS that removes undesired cells thus increasing the per cent of the desired cell population prior to FACS. FACS would then lead to a higher percent purity of the desired cells. As important, the desired pre-step procedure must leave the desired cell population at close to 80-100% yield. The present invention solves the problem by depleting the undesired cells leaving the desired cells in very high yield (Fig 1; right column; RBI is Russell Biotech, Inc.) so that FACS can be used to sort the desired cells to much higher purity while reducing the sorting time. Summary of the Invention:

The invention provides means to improve FACS so that one can obtain purer cell populations in must faster times thus solving a major problem with FACS especially when the desired cell population represents a low percentage of the starting cell population. A preferred embodiment uses metal magnetic particles as described in US Patent 9,435,799 and US Patent 9,739,768 incorporated herein by reference. The unique features of the particles: 1. rapid magnetic separation times, 2. rapid mixing times and 3. the ability to work directly in un diluted whole blood with minimal loss of non-targeted cells solve the problem at hand— yielding a desired cell population in very high yield so that in combination with FACS leads to a very high final purity of the desired cell population and in much shorter times than possible if the invention we not used.

Another embodiment uses dense particles as described in US patent 5576185 incorporated herein by reference. Though larger than particles in‘799 thus exhibiting slower reaction times the particles can be used to bind to and remove undesired cells yielding a desired cell population in very high yield prior to FACS. Brief Descriptions of Drawings:

Figure 1. Use of dense metallic nickel magnetic particles of the invention compared to use of two separate products based on iron oxide superparamagnetic particles of the art (taken from BD Biosciences catalog). It is clear that particles of the invention yield higher recoveries than particles of the art.

Figure 2. Use of metallic nickel magnetic particles of the invention to demonstrate the rapid reaction kinetics seen with these particles. Anti-CDl5 was bound to lmicron metallic nickel magnetic particles and used to determine how rapidly CD 15+ cells could be removed from whole blood. The whole blood was treated with the CD 15+ particles for the times indicated, the tube was placed in a magnetic field for one minute. The sample was then removed from the tube (while still in the magnetic field) and analyzed on a Coulter 3part diff hematology analyzer. Left peak is lymphocytes; the middle peak is monocytes and the right peak is granulocytes that are positive for CD15.

Figure 3. Demonstrates removal of specific cell populations using magnetic particles of the invention while leaving non-targeted cells in extremely high recovery. The top figure represents PBMCs; bottom box: lymphocytes; middle box: monocytes; top box granulocytes. The sample was run on a BD Flow Cytometer. Parameters measured: forward light scatter and 90degree light scatter. The middle figure shows the results after the sample was treated with CD 15+ magnetic particles of the invention. The bottom figure shows depletion of all leukocytes (lymphocytes, monocytes and granulocytes) with a combination of separate magnetic particles of the invention bound with anti-CD45, anti-CD4 and anti-CD 15 monoclonal antibodies. The small depletion of monocytes (17.5%; middle figure) is specific depletion as it is known in the art that a subset of monocytes have CD 15 on their surface.

Figure 4. Demonstrates high recovery of very rare circulating prostate cancer cells following partial depletion of CD45+ leukocytes from whole blood from a clinical metastatic prostate cancer patient. Tumor cells were determined using a circulating tumor cell assay developed by Immunicon Corp (reference 9). Figure 5. Demonstrates rapid removal of unwanted cells with high recovery of non-targeted cells using dense particles of the invention and gravity settling. Granulocytes (CD15+), monocytes (CD 15+ and CD 14+) and platelets(CD4l+) were depleted. Samples were analyzed on a Coulter STKS hematology analyzer.

Figure 6. Enrichment of CD34+ stems cells from growth factor stimulated leukapheresed peripheral blood. CD34+ cells were enriched by depleting CD15+, CD2+, CD19+, CD4+, CD8+ and CD14+ cells using dense particles of the invention. Results were obtained on a Coulter Flow Cytometer.

Detailed Description of Preferred Embodiments

The preferred means to obtain highly enriched desired cell populations in high yield prior to FACS as detailed in this invention comprise two separate methods all based on

nano/microparticle technology. The methods include: 1. metallic magnetic particles whereby undesired cells are removed by a magnetic field and 2. Dense particles whereby the undesired cells are removed by simple gravity sedimentation or accelerated gravity sedimentation

(centrifugation). In all of the methods of the invention the particles have bound thereto antibodies that are specific to the undesired cell populations to be removed prior to FACS. The metallic magnetic particles are made of magnetic metals, such as nickel, cobalt and iron but not limited to, and/or magnetic metal alloys. The dense particles are made of metals/alloys, but not limited to, that are sufficiently dense, i.e. greater than 6gm/cm3, to settle by gravity.

The antibodies used in this invention can be either polyclonal or monoclonal. Such monoclonal antibodies (Mab) are commercially available from a number of suppliers. Mab are coupled to the particles of the invention by means known in the art including adsorption and numerous covalent coupling procedures. The Mab can also be coupled to the particle using the

biotin/streptavidin/avidin system as known in the art where usually the streptavidin/avidin is bound to the particle and the antibody is coupled to biotin. Other coupling pairs known in the art are included in the invention. The sample, not limited to, can be whole blood (diluted or un-diluted), peripheral blood mononuclear cells (PMBC), usually obtained by centrifugation over Ficoll, buffy coat, bone marrow and vertebral body bone marrow. Whole blood samples can be human or animal such as mouse or rat.

The preferred embodiment uses a dense magnetic metallic particle as described in Russell‘799. The particle can range in diameter from 0.1 micron to 3.5 micron with a preferred diameter around 1 micron. The particle is dense having a density of 6-10 g/cc with a preferred density around 9 g/cc. Any metal or metal alloy that is magnetic will function in the invention with a preferred metal being nickel. Any magnetic metal or alloy commercially available that has the properties identified here such as from Sigma Aldrich or Novamet but not limited to will function in the assay but the preferred nickel particle is that described in Russell‘799.

In the preferred method, the sample will be mixed directly with magnetic nickel particles bound with antibodies that bind to the cells to be removed by end-over-end mixing

(www.atrbiotech.com /benchtop/rotomix.htm) for volumes greater than 1 ml and vortexing for volumes equal to or less than 1 ml for the shortest time possible for binding to the cells in the range of a few seconds to 10 minutes but not limited to. For end-over-end mixing the preferred rotation speed is around 15-30 rpm for 1 micron nickel particles. Following mixing the sample will be placed in a magnetic field depending on the sample volume for a few seconds up to 5 minutes. Suitable magnets are known in the art such as those from Dexter Magnetics

(www.dexteimag.com/products/medical-solutions/biomagnetic -separators-magnetic-bead- separation). The whole blood sample depleted of targeted cells will be removed while the sample chamber remains in the magnetic field and then prepared for cell sorting (FACS) using known procedures/instruments known in the art provided by manufacturers but not limited to BD Biosciences and Beckman Coulter.

As mentioned here any procedure known in the art for coupling antibody to the particles is incorporated herein. Though experimental studies may be needed to obtain the optimal amount of antibody per particle routinely labelling at 2mg antibody/meter squared particle surface (surface area) is usually sufficient. It is to be appreciated that the method shown here is a general method and it is to be expected that investigators will experimentally determine the desired particles, incubation time, volume and magnetic separation time for each desired experiment in order to best prepare the desired depleted and highly enriched sample prior to FACS.

The General Method for Depletion is detailed here:

1. Rinse the desired particles as described in Russell‘799 prior to use

2. Add particles directly to sample

3. Mix by vortexing or end-over-end mixing for volumes greater than lml for usually 1-4 minutes; Mix by vortexing for volumes less than lml; actual mixing time is determined by experimentation.

4. Immediately place in a magnetic field for a few seconds to a minute; actual time is

determined by experimentation

5. Remove the sample while it is still in the magnetic field and prepare the sample for FACS by standard means know in the art.

The use of this preferred embodiment of the invention to rapidly deplete cell populations and yield a desired cell population in high yield as a pre-step for FACS will be described in the following examples, which are intended to be illustrative of the invention, but in no way limiting of its scope.

EXAMPLE 1

Granulocytes are rapidly removed from a whole blood sample

Figure 2 demonstrates the ability to remove granulocytes from l50ul of an undiluted whole blood sample very rapidly. The control sample (Top Left) was run on a 3part differential hematology analyzer revealing three populations: lymphocytes, monocytes and granulocytes. CD 15+ granulocytes were removed with very rapid mixing times from 1 minute down to only 3 seconds. EXAMPLE 2

ETnprecedented recovery of a desired cell population

in a pre-step prior to cell sorting (FACS)

Table 1 shows that CD 15 particles of the invention can remove granulocytes to un-detectable levels while lymphocytes are recovered from 94-100 percent. Magnetic particles of the art only yield recoveries in 60-70 percent range (Figure 1). Table 1 : Rapid Removal of Targeted Cells with Very High Recovery of Desired Cells

*Time whole blood was incubated with CD 15

#Not Detectable Figure 3 demonstrates removal of various cell populations using particles of the invention showing again that cells can be depleted greater than 99% while sparing desired cells and yielding the desired cell population at 100% in this example (middle figure in fig 3 showing 99.1% depletion of granulocytes with 100% recovery of lymphocytes). EXAMPLE 3

Even cells as rare as prostate cancer cells circulating in peripheral

are enriched in high yield

CD45+ particles were used to partially deplete leukocytes from a whole blood sample from a prostate cancer patient and to determine if circulating tumor cells were recovered in high yield. The answer is yes (Figure 4). It is clear from these results that cells present at 35-40 cells per 2ml versus leukocytes at -10,000,000 cells per 2ml are spared and recovered in very high yield when using particles of the invention.

It is clear that magnetic particles of the invention meet the requirements of a pre-step prior to FACS and meet an unmet need in the field of FACS: enrichment of a desired cell population in very high yield so that following FACS the desired cell population can be sorted to very high purity and rapidly.

A second embodiment uses dense particles such as those described in U S Patent 5,576,185 incorporated herein by reference and sold by Novamet. The particles are dense with densities in the range 5-10 g/cc with a preferred density around 9g/cc. The particles can be any metal, metal alloy or ceramic that can settle by gravity in whole blood or PBMC. The preferred particle diameter is 3-10 micron. Though any diameter particle that operates as described herein is covered by the invention. The method involves dense particles with antibodies bound thereto that bind to undesired cell populations. The particles can be added directly to whole blood or whole blood can be added to particles that are at the bottom of the mixing container. The particles are mixed by end-over-end mixing at about 5-10 rpm for volumes greater than lml and by vortexing for volumes of 1 ml or less. Following the mixing step, the sample container is simply placed in an upright position and the dense particles settle by gravity to the bottom of the tube. The gravity settling can be accelerated by centrifugation at speeds that settle the dense particles but do not significantly settle the cells present in whole blood. Depending on the sample volume settling times can range from 1-10 minutes. For dense particles that are also magnetic, such as nickel particles, a magnet can be placed at the bottom of the tube following gravity settling to hold the particles in place while the supernatant is removed and prepared for FACS.

The use of an embodiment of the invention to rapidly deplete cell populations by gravity settling and yield a desired cell population in high yield as a pre-step for FACS will be described in the following examples, which are intended to be illustrative of the invention, but in no way limiting of its scope.

Example 4

Dense particles of the invention (Figure 5) (3micron nickel particles) were used to enrich lymphocytes directly from whole blood by removing granulocytes, platelets and monocytes. Note that even while treating with 3 different dense particles lymphocytes are enriched with 87% recovery a yield sufficiently high to then be used to purify lymphocyte subsets by FACS.

Example 5

CD34+ stem cells are present at a low percent in bone marrow (left figure in figure 6) of around 3.7%. ETsing dense particles of the invention to remove CD 15+ granulocytes and CD2+, CD4+, CD8+ and CD19+ lymphocytes the CD34+ cells are enriched to 35.9% and obtained in very high yield of 94%. The enriched CD34+stem cells can then be purified further using FACS.

Kits will be provided to the market for using the technology described in this invention to solve a major problem faced by researchers that of obtaining a highly enriched desired cell population in high yield prior to further purification using FACS. The kits will include both consumable reagents and equipment. The consumables will be particles (magnetic or dense magnetic/non magnetic) of the invention in a suitable buffer such as Phosphate Buffered Saline (PBS) with protein such as 0.1% BSA, but not limited thereto. The particles will have the appropriate antibody(s) bound thereto as described in the preferred embodiments. It is anticipated the particles will be bottled as 2ml and 5ml solutions but not limited thereto. The kit will also contain a suitable rinsing buffer known in the art such as PBS with 0.1% BSA. The equipment will be for a mixing means (www.atrbiotech.com /benchtop/rotomix.htm) and for magnetic particles a magnetic separation means (www.dextermag.com/products/medical- solutions/biomagnetic-separators-magnetic-bead-separation).

While the present invention has been described in terms of its preferred embodiments, it is to be appreciated that the invention is not limited thereby, and that one skilled in the art can conceive of numerous variations and modifications of the invention described herein, without departing from the spirit and scope of following claims.

Cited References:

Patents/Patent Applications:

5,576,185 Coulter Nov. 19, 1996

9,435,799 Russell Sept. 6, 2016

5,466,574 Liberti Nov. 4, 1995

5,411,863 Miltenyi May 2, 1995

4,654,267 Ugelstad Mar. 31, 1987

9,739,768 Russell August 22, 2017

4,707,523 Chang Nov. 17, 1987

Other Publications:

1. Herzenberg, H.A. etal; The history and future of fluorescence activated cell sorter and flow cytometry: a view from Stanford. Clin Chem vol 10; p 1819; 2002.

2. Ibrahim, S.F. and van den Engh, G; Flow Cytometry and cell sorting. Adv Biochem Eng Biotechnology vol 106; p 19; 2007.

3. Herzenberg, H.A, etal; Fetal cells in blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. PNAS vol 76; p 1453 1979.

4. Pruzak, J. etal. Markers and methods for cell sorting of human embryonic stem cell derived neural cell populations. Vol 25; p 2257; 2007.

5.Mattanovich, D. and Borth, N. Applications of cell sorting in biotechnology. Microbial cell factories; vol 5; 2006.

6. Cell Separation Methods and Applications; edited by Recktenwald and Radbruch; Publisher: Marcel Dekker, Inc. 1998. 7. Examples of product catalogs referencing cell sorting by Fluorescence Activated Cell Sorting (FACS): BD Biosciences and Beckman Coulter 8. Examples of Product Catalogs: Miltenyi Biotec; BD Biosciences; EMD Millipore; Stem Cell technologies; Beckman Coulter, PolySciences.

9. Kagan M, Howard D, Bendele T, Mayes J, Silvia J, Repollet M, Doyle J, Allard J, Tu N, Bui T, Russell T, Rao C, Hermann M, Rutner H, Terstappen LWMM. A Sample Preparation and Analysis System for Identification of Circulating Tumor Cells. J Clinical Ligand Assay, 25, 104- 110, 2002.