WO2020236850A1 - Solid phase devices and methods of use for purifying and quantifying tissue-specific leukocytes - Google Patents

Abstract

The present invention relates to immunotherapy, transplant therapy, therapy for rheumatic diseases, therapy for inflammatory diseases, and assessment of disease progression and immunotolerance. It provides devices that allow capturing and monitoring the number and affinity of leukocytes from a subject's blood that are specific to a tissue, cancer, or infectious disease based on selective interactions between the leukocytes and immobilized targets derived from a tissue or cancer. Further taught are methods of use of captured leukocytes in immunotherapy and the therapeutic use of quantifying such cells.

Description

SOLID PHASE DEVICES AND METHODS OF USE FOR PURIFYING AND QUANTIFYING TISSUE-SPECIFIC LEUKOCYTES

CROSS-REFERENCE TO RELATED APPLICATIONS

001 This application is claims the benefit of U.S. provisional patent application Serial No: 62/850,126 filed 20 May 2019, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

002 This present invention relates generally to the field of immunotherapy, transplantation, and treatment for cancer and other pathologic conditions.

BACKGROUND

003 Immunotherapy (e.g. adoptive T cell therapy, checkpoint blockade, therapies using tumor-infiltrating lymphocytes, T cell receptor modified T cells, and chimeric antigen receptor-T cells) has been a major advance in the therapy of melanoma and other solid malignancies such as non-small cell lung cancer (NSCLC). Although immunotherapies have demonstrated significant clinical benefit for treating some cancers in some subjects, many subjects do not clinically respond to such inhibition.

For example, only 10-20% of NSCLC patients respond. Accordingly, identifying an accurate biomarker that predicts an effective response has been the subject of intense study. Since therapies such as immune checkpoint inhibitors (e.g. anti-PD-1 , anti-PD- L1 , and anti-CTLA-4 antibodies are significantly toxic and very expensive, there is a great need in the art to identify methods that are predictive of patient responsiveness to immunotherapies in order to appropriately determine an efficacious and cost-effective course of therapeutic intervention.

SUMMARY OF THE INVENTION

004 Provided here are devices and methods of their use that allow capturing and monitoring the number of leukocytes from a patient’s blood that recognize a patient’s tumor based on selective interactions between immune cells and immobilized targets derived from the patient’s cancer. Such capturing provides a bases of harvesting such leukocytes for immunotherapy. Such monitoring allows dynamic assessment of immunotherapy efficacy and provides valuable information to the physician to alter or continue therapy to reach superior outcomes. 005 The core device of the present invention comprises a vessel (i.e. inanimate container) with a lumen, the lumen comprising a substrate, and the substrate being attached with an MHC binding agent. Typically, the MHC binding agent is optionally attached by means of a tether wherein the tether is attached to the substrate by the tether’s proximal end, and the tether being attached at its distal end with the MHC binding agent.

006 The core device of the present invention is activated (to form an activated device) by binding patient peptide-MHC complexes to the substrate (i.e. through the MHC-binding agent).

007 The reporter device of the present invention is the activated device to which leukocytes (e.g., T cells) have been passed over and the tissue-specific (e.g. cancer- specific or infection-specific or organ specific) cells have been allowed to bind to the peptide-MHC complexes (e.g. through cell receptors such as the T cell receptor). Immunocompetence (defined below) is determined by first quantifying the number of bound leukocytes per unit area of the membrane and then back calculating the number of bound leukocytes per unit volume of blood.

008 With the present invention, it is now possible to quantify tissue-specific (e.g. infection-specific or cancer-specific or organ specific) leukocytes without the need for biochemical, genetic, or immunologic characterization

009 In one embodiment, in the instant reporter device, the vessel is glass, the substrate is partitioned into microfluidic channels, the MHC-binding agent is an anti- MHC antibody, the peptide-MHC complex is derived from a cancer biopsy from a subject, the tether comprises polydimethylsiloxane polymer, the anti-MHC antibody is bound to the tether through a biotin-streptavidin bridge, the leukocytes are cytotoxic T- cells from the subject and bound to the peptide-MHC complex through the T-cell receptor, and the device is a closed vessel further comprising a pump.

0010 In one embodiment, the present invention comprises a method of enriching a population of tissue-specific leukocytes (e.g. cancer-specific or infection specific or organ leukocytes). In another embodiment, the present invention comprises a method of quantifying leukocytes reactive with a subject’s cancer (i.e.“Immunocompetence”).

In another embodiment, the present invention comprises a prognostic method. In another embodiment, the present invention comprises quantifying a patient’s Immunocompetence relative to the patient’s cancer (as taught below) at a first point in time, administering an immune checkpoint inhibitor or other immunotherapy, quantifying the patient’s Immunocompetence (relative to the patient’s cancer) at a second point in time (e.g. days or weeks after the first time point), and continuing or ceasing administration of the immunotherapy depending upon whether the patients’ Immunocompetence increases or decreases (respectively).

001 1 In one embodiment, the present invention is a kit comprising core device (or a core device but with the MHC-binding agent provided in a vessel in the kit) and directions for use comprising instructions for making the activated device and the reporter device. The directions optionally contain instructions for one or more of (i) making peptide-MHC-complex preparation; making the leukocyte fraction; quantifying the bound leukocytes.

0012 BRIEF DESCRIPTION OF THE DRAWINGS

0013 Figure 1 shows a schematic of one embodiment of an instant core device.

0014 Figure 2 shows a schematic of one embodiment of an instant activated device.

0015 Figure 3 shows a schematic of one embodiment of an instant reporter device.

0016 Figure 4 is a phase contrast micrograph of captured leukocytes.

0017 Figure 5 is a bar graph showing the percentage of adherent cells in the citing Example.

0018 Figure 6 is a bar graph showing the effect of sheer force on adherence of control leukocytes.

0019 Figure 7 is a fluorescence micrograph of captured primary human T-cells

DETAILED DESCRIPTION OF THE INVENTION

0020 As used here, the following definitions and abbreviations apply:

0021 “Cancer-specific”, in the context of leukocytes means leukocytes which are capable of binding to cancer tissue with an affinity higher than to non-cancer tissue.

0022 “Examplary” (or“e.g.” or“by example”) means a non-limiting example.

0023 “Immunocompetence”, as used herein, refers generally to a subject’s ability to produce leukocytes specific for a peptide-MHC complex species. It can be expressed as the number of tissue-specific or cancer-specific leukocytes as measured according to the present invention per unit volume of blood or total leukocytes.

Applicant recognizes that this usage is a somewhat different than more common used and thus the Applicant uses it herein in the first letter uppercase form.

0024 “MHC” means major histocompatibility complex. As used herein, MHC typically, but not exclusively, refers to MHC class I.

0025 “Specific binding”, in reference to leuckocytes specifically binding to the instant device means binding through the peptide-MHC complex at an infinity greater than that of a leukocyte lacking the receptor for the peptide-MHC complex.

0026 “Subject” means an individual animal (e.g. human, primate, rodent, companion animals, equine, food production animals, etc.).

0027 “Tissue-specific”, in the context of leukocytes means leukocytes which are capable of binding to a specified tissue or a tissue of one subject with an affinity higher than to tissues other than the specified tissue or with an affinity higher than to a tissue from the same organ but from a different subject.

Devices

Core Device

0028 The core device comprises a vessel, a substrate, and a MHC-binding agent attached to the substrate.

0029 In one embodiment, the MHC-binding agent is attached to the substrate by a tether.

0030 The vessel of the core device can be a closed vessel comprising two ports useful for introducing fluid or fluid containing samples into (or out of) the vessel.

0031 The vessel of the core device can be a vessel with a reversible closure.

0032 Additionally, the core devices can further comprise a pump to deliver fluids or fluids containing biological samples into contact with the substrate or components tethered thereto and out of the vessel.

0033 The substrate can additionally be partitioned into compartments.

Activated Device 0034 The core device of the present invention is activated by passing peptide-MHC complex -enriched preparations through the device under conditions that allow patient peptide-MHC complexes to bind to the MHC binding agent. Typically, this preparation is made by obtaining relevant tissue from the subject (e.g. from a biopsy such as a tumor biopsy), lysing the cells, solubilizing the membrane proteins, and removing organelles by ultracentrifugation.

Reporter Device

0035 The activated device becomes a reporter device of the present invention by the addition of a sample of a subject’s blood (or cells derived from a subject’s blood resuspended in a buffer). The read-out of the reporter device is the number of leukocytes bound to the membrane per unit area of membrane - quantified by any means known to the skilled artisan (e.g. cell count, immunoassay, etc.).

Vessel

0036 With the teaching herein, the skilled artisan will readily recognize the wide variety of vessels that can be used, dependent upon the embodiment. The vessel only requires that it has a shape that can contain fluid, e.g. a rectangular prism, a triangular prism, a cylinder, a sphere, a tetrahedron, and curved versions of the same. For example, when the substrate is glass beads, the vessels can be 1.5 ml polypropylene microcentrifuge tubes, 15 ml conical centrifuge tubes (polyethylene or polypropylene), open glass test tubes, cylindrical tubes (e.g. a used for column chromatography) and the like.

0037 When the substrate is a quasi-planar surface, suitable vessels can be cuboidal chambers with an opening for access or with two ports (e.g. inlet and outlet) or a flat bottom flask with a screw-cap neck.

0038 The vessel can be treated by various known methods of blocking non-specific binding (e.g. coating with protein such as albumin or milk proteins). Non-specific binding can also be reduced by increasing fluid flow rate to increase shear force.

Substrate

0039 Substrates useful according to the present invention are any substrates known by the skilled artisan to be capable of binding a tether or the MHC-binding agent directly. 0040 Suitable substrate materials include solids such as glass, silica, polydimethylsiloxane, plastic, metal, fiber, latex, heavily cross-linked polystyrene or similar polymers, gold or other colloidal metal particles. In some embodiments, such substrates can be derivatized with chemical groups suitable for binding to the MHC- binding agent.

0041 Examples of suitable polymer materials are latex, rubber, polyethylene, polypropylene, polystyrene, a styrene-butadiene copolymer, polyvinyl chloride, polyvinyl acetate, polyacrylamide, polymethacrylate, a styrene-methacrylate

copolymer, polyglycidyl methacrylate, an acrolein-ethylene glycol dimethacrylate copolymer, polyvinylidene difluoride (PVDF), silicone, agarose, and gelatin.

0042 Suitable substrates include magnetic particles (or magnetic bodies), which can be separated from fluids by placing them in a magnetic field.

0043 Where the substrate is magnetic particles, the particles can contain a magnetic material as the base material. Such magnetic particles are known in the art and include, for example, particles using Fe₂C>3 and/or Fe₃C>4, cobalt, nickel, ferrite and magnetite as the base material. For the purpose of binding an anti-MHC protein antibody, etc. onto the surfaces of magnetic particles, the base material is preferably coated thereon with a polymer etc.

0044 In one embodiment, the substrate is made of magnetic glass as set forth in United States Patent 4297337.

0045 In one embodiment, the substrate is a negatively charged polymeric material with a coating of polyethyleneimine.

0046 According to another embodiment, the substrate is a carboxylate-modified latex with a coating of polyethyleneimine.

0047 In yet another embodiment, the substrate is a solid support containing a layer of microparticles coated on the solid support, the microparticles being formed of a negatively charged polymeric material; and a coating of polyethyleneimine on the microparticles.

0048 In another embodiment, the substrate comprises a negatively charged polymeric material; a coating of polyethyleneimine; and an immunoreagent immobilized on the substrate by covalent coupling to the polyethyleneimine coating (directly or through a tether).

0049 In another embodiment, the substrate is coated with polyethyleneimine to inhibit contact activation of plasma brought in contact with the surface.

0050 According to another embodiment, the substrate comprises microparticles comprising a carboxy-modified latex; a coating of polyethyleneimine on the

microparticles; and an anti-MHC protein antibody immobilized on the microparticles by covalent coupling to the said polyethyleneimine coating (directly or through a tether).

0051 Monobeads™ (10 pm) (Pharmacia Fine Chemicals AB, Uppsala Sweden) or their equivalent, are useful as substrates.

0052 In one embodiment, the substrate is polystyrene beads, polystyrene tubes, or a polystyrene quasi-planar surface. By way of example, anti- MHC antibodies can be coated to the polystyrene substrate (directly or by way of a tether) by any method known to the skilled artisan, such as set forth in United States Patent 4693969.

0053 In another embodiment, the substrate is affixed to the vessel.

0054 In another embodiment, the substrate is one or more luminal surfaces of the vessel.

0055 It has been discovered that techniques useful for immunoaffinity purification can be adapted to the methods of the present invention. By way of examples, the skilled artisan will now recognize that many of the methods taught in Methods in Enzymology Volume 559, 2015, Pages 27-36, Chapter Three - Immunoaffinity

Purification of Proteins (Jennifer M.Kavran and Daniel J. Leahy) are useful.

0056 In one embodiment, anti-MHC antibody is covalently linked to

aminopolystyrene using dimethyl suberimidate as taught in US Pat No. 5525473, Optionally, the polystyrene is in the form of a macro-bead, e.g. 5/16 inch diameter.

0057 These materials may be used alone or as a mixture of two or more thereof.

Micro-compartments

0058 In some embodiments, the substrate is partitioned into micro-compartments (or channels or microfluidic channels). In some embodiments, the micro-compartments can be formed using micro-fabrication techniques such as photolithography, acid etching, soft lithography, or drilling as taught in US 8,753,309 B2. In some

embodiments, the substrate is glass and partitioned into micro-compartments.

Surface area

0059 With the teaching herein, the skilled artisan will now recognize the amount of substrate surface area that is required to generate meaningful signals (i.e. to quantify the specific leukocytes in a specimen). By way of example, useful surface areas are 1 mm² to 100 cm².

Pump and Shear Force

0060 In one embodiment, the leukocytes are pumped continuously through the vessel over the substrate. Using COMSOL multiphysics software or other appropriate software, the fluid flow and shear stress passing over the substrate (e.g. within a channel) can be estimated and the pump rate adjusted to obtain optimal leukocyte binding (e.g. of lymphocytes specific for tumor peptide antigens). By adjusting the flow rate, it has been discovered by Applicants that non-specific binding of leukocytes can be reduced as shown in Example 5.

0061 It has also been discovered that leukocytes bound specifically in the instant device can be characterized by affinity to the peptide-MHC complex according to the shear force that can be applied without dissociating the leukocytes. By way of example, leukocytes can be added at a shear force previously determined to be sufficient to prevent non-specific binding. Next, the shear force can be increased (in steps or in a gradient) and disassociated leukocytes can be collected and quantified. The, leukocytes can be fractionated according to affinity to the tumor peptide antigen.

In the mind of the inventor, certain fractions are more diagnostic of a patient’s response to immunotherapy.

0062 Alternatively, leukocytes can be pumped through the vessel at a range of shear stresses and the number of adherent leukocytes can be characterized based upon the shear stress against which the cells attach.

0063 Alternatively, subject leukocytes can be characterized by microvideography and observing how they attach and detach to the device under various shear forces.

MHC binding agent 0064 The MHC binding agent, according to the present invention, can be any agent known to bind MHC Class 1 type. For example, any polyclonal or monoclonal antibodies recognizing MHC proteins are useful provided that they bind native MHC and do not block the tumor antigen binding site or prevent the complex from binding to T cell receptor. By way of example, Invitrogen sells more than 50 different monoclonal antibodies that react with human MHC Class 1 proteins.

0065 In one embodiment, the anti-MHC antibody is a biotinylated antibody and the tether comprises an avidin or streptavidin molecule, thus facilitating the binding of the biotinylated antibody to the avidinylated or streptavidinylated tether.

0066 In another embodiment, the tether comprises Protein A, Protein G, or Protein A/G (a chimeric fusion protein).

0067 It had been discovered, in the mind of the inventor, that optimized

concentrations of MHC binding agent are important to increase the signal to noise ratio in the read-out step.

Tethers

0068 It has been discovered in the mind of the inventor that by adding a tether between the substrate and the MHC binding agent, the peptide-MHC complex is better accessible to the MHC binding agent, and the peptide-MHC complex is better able to bind leukocytes.

0069 For example, a useful tether is longer than about 10 angstroms or longer than 20 angstroms or longer than 40 angstroms.

0070 In certain embodiments, the tether comprises a nanopolymer. In certain embodiments, the tether is selected from the group consisting of polylysine, polyglutamic acid, N-(2-hydroxypropyflmethacrylamide, polycation polymers, poly(allylamine), poly(dimethyldiallyammonim chloride) polylysine, poly(ethylenimine), poly(allylamine), natural polycations, dextran amine, polyarginine, chitosan, gelatine A, protamine sulfate, polyanion polymers, poly(styrenesulfonate), polyglutamic or alginic acids, poly(acrylic acid), poly(aspartic acid), poly(glutaric acid), natural polyelectrolytes with similar ionized groups, dextran sulfate, carboxymethyl cellulose, hyaluronic acid, sodium alginate, gelatin B, chondroitin sulfate, and heparin.

0071 In one embodiment, the tether is made up of a polydimethylsiloxane polymer. Cleavable tether

0072 In one embodiment, the tether is a cleavable tether, for example, a photo- cleavable tether. In some embodiments, the photocleavable linkers are 6- nitroveratryloxycarbonyl (NVOC) and other NVOC related linker compounds (see WO 90/15070 and WO 92/10092; U.S. Ser. No. 07/971 ,181 , filed Nov. 2, 1992). Other suitable linkers include nucleic acids with one or more restriction sites, or peptides with protease cleavage sites (see, e.g., U.S. Pat. No. 5,382,513). The cleavable tether allows the release of the T-Cells for convenience of quantifying the amounts.

Read-out / Reporter

0073 Leukocytes attached to the activated device (through the peptide -MHO complex bound to the MHO binding agent bound directly or through a tether to the substrate) can be quantified by any means known to the skilled artisan.

0074 By way of example, the leukocytes can be counted under suitable microscopy such as phase contrast microscopy. In another embodiment, the leukocyte can be quantified by immuno-assay (e.g. immunofluorescence). In yet another embodiment, the leukocytes can be labeled with fluorescent dye and counted using fluorescent microscopy.

0075 In one embodiment, the leukocytes can be released by any methods known in the art and then the leukocytes are ounted by any method known to the skilled artisan.

0076 By way of example, the leukocytes can be released by proteolytic cleavage (e.g. trypsin or papain) in a manner that cleaves the peptide-MHC complex or the peptide-MHC complex-binding site on the leukocytes or the MHC binding agent.

0077 In another embodiment, the leukocytes can be released by pumping fluid through the vessel at such a rate as to create shear force sufficient to release them and then quantifying the released leukocytes by standard techniques known in the art

0078 By way of example, the leukocytes can be counted under suitable

microscopy such as phase contrast microscopy. By way of example, the leukocytes can be counted under suitable microscopy such as phase contrast microscopy.

0079 In another embodiment, the leukocytes can be quantified by an immuno- florescence assay. 0080 In another embodiment, the leukocytes are qualitatively analyzed as a more refined indicator of Immunocompetence.

0081 For example, leukocytes can also be analyzed for CD8 expression, as an indicator of cytotoxic T cells.

0082 For example, PD-1 expression can be analyzed (e.g. as an indicator of T-cell exhaustion).

0083 Similarly, leukocytes can also be characterized for expression of granzyme B, CD4 (as an indicator of helper T lymphocytes), CD8 (as an indicator of cytotoxic T lymphocytes), CD19 (as an indicator of B-lymphocytes), CD15 (as an indicator of neutrophils), FoxP3 (as an indicator of regulatory T lymphocytes), CD68 (as an indicator of macrophages), S-100, PD-L1.

0084 Leukocyte dysfunction can also be analyzed by expression of CD27^hl, CD28¹, °CD57^|0CD127¹⁰, CCR7, CD45RA, CD27, and CD45RO.

Peptide-MHC complexes

0085 Peptide-MHC complexes are obtained from a subject biopsy. The skilled artisan will now readily appreciate a variety of useful methods. Crude extracts can be used or peptide-MHC complexes can be partially purified by sizing (e.g. gel filtration) or immunoaffinity methods.

0086 Peptide-MHC complexes can be obtained as set forth in Example 2.

0087 In another embodiment, peptide-MHC complexes are prepared by the method of Altman et al. (Science 1996, 274: 94-96).

0088 In another embodiment, peptide-MHC complexes are prepared by the method of Sidney et al. (Curr Protoc Immunol. 2013 100:18.3.1 -18.3.36).

0089 In another embodiment, peptide-MHC complexes are prepared by the method of Malik and Strominger (Journal of Immunological Methods 234, Issues 1-2, 3

February 2000, Pages 83-88).

0090 In one embodiment, the tissue biopsy is washed in cold PBS, minced, and lysed in lysis buffer for 45 min at 4L C on a rotator. The suspension is centrifuged at 20,000g, 4LC for 20 minutes. Next, the supernatant is incubated with anti-MHC antibody and incubated at 4LC on a rotator overnight. Next, protein G-magnetic beads are incubated with CHAPS buffer (500 pg) at 4LC on a rotator for 2 hr followed by washing 4 times with CHAPS buffer. The peptide-MHC complexes are released from the immunomagnetic beads by acidic pH and/or hyperosmolarity.

Leukocyte preparations

0091 The skilled artisan will now readily appreciate a variety of useful methods of obtaining leukocyte preparations for passing over the instant activated device. For example, whole blood can be passed over the instant activated device. Alternatively, such preparation can first be partially purified by lysing red blood cells (e.g. by hypo- osmotic shock followed by restoring isotonicity). Further purification can be obtained by centrifuging whole blood and aspirating the buffy coat (and resuspending it in an isotonic, neutral buffered solution). Further purification can be obtained by density gradient centrifugation (e.g. in ficoll hypaque as set forth in Example 3). Leukocytes can also be prepared by immunoaffinity purification (e.g. anti-CD8) or rapidly selected by flow cytometry. Leukocytes can include any of the cells typically found in the buffy coat following centrifugation of whole blood (e.g., lymphocytes, natural killer cells, monocytes).

Method of Manufacture

The device of the present invention is made by the steps shown below, wherein each step is more fully described above:

0092 A vessel comprising a substrate on luminal side is made;

0093 an MHC binding agent is tethered to the substrate;

0094 a biopsy from a tissue (e.g. cancerous tissue) of a subject is obtained;

0095 the biopsy is processed to produce a preparation of soluble peptide-MHC complexes;

0096 the preparation is incubated in the lumen of the vessel for a time sufficient for the peptide-MHC complexes to bind to the anti-MHC antibody;

0097 the lumen of the vessel is washed to remove components of the preparation other than the peptide-MHC complexes;

0098 a leukocyte sample is obtained from the subject; 0099 the leukocyte sample is incubated in the lumen of the vessel for a time sufficient for cancer-specific leukocytes to bind to the peptide-MHC complexes; and

00100 the lumen of the vessel is washed to remove components of the leukocyte sample other than the cancer-specific leukocytes.

00101 In another embodiment, the device of the present invention is made by the following steps:

00102 A vessel comprising a substrate on luminal side is made;

00103 an MHC binding agent is tethered to the substrate;

00104 a biopsy from a tissue of a subject is obtained;

00105 the biopsy is processed to produce a preparation of soluble peptide-MHC complexes;

00106 a leukocyte sample is obtained from the subject;

00107 the leukocyte sample is incubated with the biopsy preparation for a time sufficient for the cancer-specific leukocytes to bind to the peptide-MHC complexes; and

00108 the solution of step f. is incubated in the lumen of the vessel for a time sufficient for the peptide-MHC complexes to bind to the MHC binding agent;; and

00109 the lumen is washed to remove components other than those bound through the MHC binding agent.

Method of obtaining a preparation of enriched tissue-specific or cancer-specific leukocytes

001 10 The skilled artisan should readily recognize that the methods of obtaining preparation of enriched tissue-specific or cancer-specific leukocytes are accomplished at the last stage of the method of manufacturing the device of the present invention, as set forth above.

001 1 1 The cancer-specific leukocytes can further be released by methods generally known to the skilled artisan. Moreover, shear force in washing, as taught above, is also a preferred method.

Utility of Enriched Leukocyte preparations 001 12 Enriched leukocyte preparations, as taught herein, can be used in any way known to the skilled artisan. By way of example, such preparations can be used for adoptive T cell therapy. In some cases, such T cells can be expanded ex vivo and then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor. Also, by example, allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes can be used to treat positive post

transplantation lymphoproliferative disease. Enriched leukocyte preparations, as taught herein, can also be used for chimeric antigen receptor (CAR) T-cell therapy.

Unexpected Results and Uses

001 13 Through insight in the mind of the inventors, difficult challenges of

immunotherapies have been solved. It is only now possible to predict the success of immune modulating therapy (e.g. checkpoint blockade). It is only now possible to identify the non-responders to immunotherapies after only a few weeks of treatment and then to initiate alternative treatment, eliminating toxicity in a non-productive treatment as well as reducing wasted financial resources. It is only now possible monitor continued success of checkpoint blockade and to effectively prolong treatment. It is only now possible to characterize the affinity of leukocytes for a subject’s peptide- MHC complex, which significance is only now being recognized.

001 14 The present invention allows the user to collect and or quantify clinically relevant information concerning cancer-specific leukocytes without molecular identification or characterization (so-called“agnostic: methodology).

001 15 The present invention allows the user to test compatibility of a donor organ with a recipient where the leukocytes are from the recipient and the peptide-MHC complexes are from the donor organ.

001 16 The present invention now provides a powerful tool to assess the status of and the progress of therapy for inflammatory diseases and infectious diseases. Examples of such inflammatory diseases that can be assessed include autoimmune diseases such as coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel disease, Crohn’s Disease, and the like. Such inflammatory diseases can include rheumatic diseases such as osteoarthritis, rheumatoid arthritis, systemic Lupus erythematosus, spondyloarthropathiesm, ankylosing spondylitis, psoriatic arthritis, Sjogren’s syndrome, gout, scleroderma, infectious arthritis, juvenile idiopathic arthritis, polymyalgia rheumatic, and the like. Examples of infectious diseases include bacterial, fungal, viral, and parasitic infectious diseases.

001 17 Assessing the status of and the progress of therapy for inflammatory and infectious diseases can now be accomplished, by way of example, by biopsying an affected region of a test subject (or an individual with the same type of inflammatory or infectious disease), a peptide-MHC complex fraction can then be prepared therefrom, and then the fraction can be incubated with the core device to make an activated device. Leukocytes would then be harvested from a test subject and then passed over the device. The binding of substantial numbers of leukocytes would be indicative of active disease and an increase or decrease in number would be indicative of poor response (or disease progression) or favorable response to therapy (or disease regression) respectively. Moreover, the specific binding of lymphocyte to the instant device can be a useful step in a method of identifying antibodies that are candidate for immunotherapy because the capture of the lymphocytes (e.g. B cells) is facilitated by the receptor which is the antibody produced thereby.The present invention now provides a powerful tool to assess and quantify immunotolerance of a subject to a viral, bacterial, or fungal disease. For this embodiment, autopsy or biopsy material from a subject with a viral, bacterial, or fungal disease is processed and added to the core device (i.e. device with anti MHC antibody at the end of the tether) to form an activated device (as described herein). Next, whites cells (crude preparations, purified lymphocytes, etc.) are collected from a test subject and loaded on to the device as described herein. Next, the white cells that are bound to the device are quantitatively analyzed by type. Such an analysis can be by the use of immunoprobes on the device or by removing the cells and quantifying them by other routine methods such as fluorescence-activated cell sorting. Quantitative assessment of certain cell types such a memory T cells can be used as a predictor of immonotollerance / resistant to the that were specific to the viral, bacterial, or fungal disease tested for.

001 18 The present invention now provides a powerful tool to collect tissue-specific leukocytes which can then be expanded ex vivo and the allogeneic or autologous transfer.

EXAMPLES

Example 1 Production of Core Device 001 19 In this example, micro-channels were fabricated in a glass vessel using photolithography. First, a mask of the device design was created. A master mold was prepared from SU8 photoresist (Microchem, MA, USA), which was coated on silicon wafer at a desirable thickness (100 -500 pm) by controlling the spin coater speed. The mask of device design and the SU8 coated wafer were contacted and exposed to UV in a mask aligner. This procedure yielded a crosslinked pattern of device design. The non-crosslinked SU8 was leached using SU8 developer and subsequently washed with isopropyl alcohol. All these procedures were carried out in a dust free clean room. The master mold was then salinized and the microdevice was created by casting polydimethylsiloxane (PDMS), which was prepared by mixing Sylgard® 184 silicone elastomer base and curing agent (both Dow Corning, Midland, Ml) in a 10 : 1 ratio, on the SU-8 master molds. The device was peeled off the master mold after heat treatment at 60 °C overnight, and then bonded to a flat PDMS sheet using air plasma. The device bonding was cured briefly at 120 °C, and devices were sterilized using UV prior to use in experiments.

00120 A glass and polydimethylsiloxane (“PDMS”) vessel of the present invention was subjected to plasma cleaning to activate the glass surface of the channels for silane binding. Immediately next, the vessel is brought to a fume hood and 100 pi of a 3-Mercaptopropyl)trimethoxysilane solution (40 mI added to 1 mil of 100% ethanol) were added to each channel and incubated at room temperature for one hour in a desiccator. After incubation each channel was washed 2 times with molecular biology grade ethanol.

00121 A N-y-maleimidobutyryl-oxysuccinimide ester (GMBS) solution was prepared by adding 2.8 mI of GMBS (4°C refrigerator) to 1 ml molecular biology grade ethanol. One hundred mI of the GMBS solution was added to each channel and incubated for 30 min at room temperature. After incubation, each channel was washed 2 times with molecular biology grade ethanol.

00122 Streptavidin solution was prepared at 100 pg/ml in phosphate buffered saline (PBS). One hundred mI of streptavidin solution was added to each channel. The solution was aspirated and another 100 mI of streptavidin solution was added. The solution was again aspirated and fresh solution was added and the vessels were stored at 4°C in wet box for up to 2-3 months. Example 2 Production of Activated Device.

00123 Cancerous or other tissue of interest is obtained from a patient biopsy, which is then minced into 1 - 2 mm pieces with a scalpel in cell lysis buffer (Radio

Immunoprecipitation Assay [RIPA]: 50mM Tris (pH 7.4), 150mM NaCI, 1 % Triton X- 100, 1 % sodium deoxycholate, 0.1 % SDS, and sodium orthovanadate, sodium fluoride, and Roche protease inhibitors Cat. No. 1 1 836 153 001 ).

00124 Next the minced tissue is subjected to mild homogenizing (e.g. dounce homogenizer) and the homogenate is subjected to centrifugation at 500xg for 10 minutes. The supernatant is placed on a rocker at 60 rpm for 24 hours. Next the suspension is centrifuged at 33,000xg for 1 minute and the supernatant is collected (“lysate”).

00125 Lysate is next incubated at 4 °C for three days with 5 pi of biotinylated anti human MHC antiserum per 100 ul lysate.

00126 Next, the lysate / antiserum is incubated in the lumen of the core device (e.g. in a micro-channels) for two days at 4 ⁰ C, followed by washing the device with lysis buffer. This is one embodiment of the“activated device”.

00127 An alternative procedure is to incubate the biotinylated anti-MHC antisera with the device, wash the device, and then to incubate the cell lysate with the device (followed by washing).

Example 3 Manufacture of the Reporter Device

00128 Venous whole blood is collected from the subject (e.g. cancer patient) in heparinized tubes, diluted 1 to 1 in phosphate buffered solution. Twenty mis of blood solution is transferred to a 50 ml conical tube and underlaid with 10 ml of histopaque 1077 and centrifuged at 700xg for 30 min at 4°C. Next, after noting the three layers, the supernatant is removed within proximity of the opaque layer (containing peripheral blood mononuclear cells; PBMCs) just above the ficoll layer. The PBMC layer is removed and transferred to a new 50 ml conical tube Q.S.’d to 45 ml with PBS, centrifuged at 500xg for 10 min at 4°C, resuspended in PBS and applied to the

Activated device. This is the“reporter device”. After washing in PBS, adherent cells are counted (e.g. by phase contrast microscopy).

Example 4 Demonstration of Utility in Mouse Model 00129 In this study, ovalbumin was used as a model for the tumor peptide antigen.

An MHC class I antigen encoded in the chicken ovalbumin gene is an 8-mer with the sequence SIINFEKL. This 8-mer binds with high affinity to the MHC class I molecule H- 2K^b. A“single chain trimer” (SCT) was created wherein the 8-mer was covalently linked to the H-2K^b a chain and b2 microglobulin. This SCT complex was then overexpressed in a murine glioblastoma cell line, GL261 , and can then readily be detected by flow cytometry using the 25-D1.16 antibody. This complex is remarkably stable and is recognized by T cells of mice who are immunized with ovalbumin or T cells of OT-1 transgenic mice in which most CD8 T cells are specific to the SIINFKEL peptide presented by H-2K^b.

00130 Next, the core device is made according to Example 1 ; that is, it is coated in streptavidin. Next, GL261 cells (those expressing the SCT complex and wild type) are lysed, the crude cell lysate is combined with a biotinylated antibody to H-2K^b or isotype control, and the lysate-antibody solution is flowed through the microfluidic channels.

The affinity of streptavidin to bind biotin allows the H-2K^b from the GL261 cells to be captured on the surface.

00131 Next, lymphocytes from the OT-1 transgenic mice or wild type mice were passed over the activated device. As shown in Figure 4, lymphocytes from sensitized mice flowing through the device were captured by time lapse recordings (left panel), demonstrating significantly enhanced capture of the lymphocytes. In contrast, the numbers of captured lymphocytes from wild type mice were substantially reduced (right panel). White arrows indicate lymphocytes flowing freely, yellow circles identify lymphocytes that have adhered to membrane (Note: shear rate is 0.01 dynes/cm²).

00132 The per cent T-cells adhering was quantified and shown in Figure 5. The first bar shows that about 60% of the T-cells from OT-1 mice bound to the device charged with MHC fraction from the SCT transformed GL261 using anti H-2K^b antibody. As shown by the second bar, only a negligible fraction of T-cells from OT-1 mice bound to the device charged with MHC fraction from the SCT transformed GL261 and control antibody. As shown by the third bar, about 30% of the T-cells from ovalbumin- immunized mice bound to the device charged with MHC fraction from wild type (not SCT transformed) GL261 cells and anti H-2K^b antibody. As shown by the fourth bar, about 30% of the T-cells from wild type mice bound to the device charged with MHC fraction from SCT transformed GL261 cells and anti H-2K^b antibody. Example 5 Decreasing Non-specific Binding Using Mouse Model

00133 In Example 4, it was noted that some non-specific binding occurred when the fluid force during the T-cell binding step was at a shear rate of 0.01 dynes/cm².

00134 This study was repeated using T-cells from OT-1 mice bound to the device charged with MHC fraction from wild type (not SCT transformed) GL261 cells and anti H-2K^b antibody. The sheer force during the T-cell loading step was increased to 0.014 dynes/cm² . As shown in Figure 6, this increase in shear force resulted in a decrease of about 50% non-specific binding.

Example 6 Demonstration of Utility Using Cultured Human Cells

00135 For this study, three different cancer models are used: melanoma cell line SK- MEL-5 which are MARTI positive, colorectal cancer cell line SW620 transfected to be MARTI positive, and breast cancer cell line MDA-MB-231 transfected to be MARTI positive. Each of these cancer cell lines expresses (i) full-length MART 1 protein (a transmembrane protein that is present in normal melanocytes and widely expressed in malignant melanoma) and (ii) Major histocompatibility complex MHCI allele for HLA- A^*0201 (human leukocyte antigen serotype; a chain), which can bind immunogenic, MARTI -derived peptide antigens.

00136 Test T-cells (positive controls) used are Jurkat cells (immortalized line of human T lymphocyte cells) which are transduced by CCLc-MND-F5 (a lentiviral vector encoding the T cell receptor (TCR) that recognizes the MART 1 peptide). Non- transformed Jurkat cells are used as negative controls.

00137 Reagents to be used include monoclonal antibodies against HLA-A2 (clone BB7.2), monoclonal antibodies against MART 1 protein (clones A103 and M2-2C10), and monoclonal antibodies against human MHC (clone W6/32). The anti-HLA-A2 and anti-MART1 antibodies are useful for immunofluorescence (e.g. on cell monolayers) or flow cytometry (performed on cell suspensions). The anti-human MHC antibody is used to purify peptide-MHC complexes from the lysate of cultured human cancer cells.

00138 MHC / tumor peptide antigen fraction is prepared from cultured test cancer cells grown to confluence on monolayers. Cells are removed with rubber scraper, treated with lysis buffer containing protease inhibitors, collected, and centrifuged at 500xg for 10 min. Supernatants are centrifuge at 100k xg and the pellet is

resuspended in suspension buffer. 00139 Cancer cell lysate is incubated with biotinylated anti-MHC antibody (or control isotype antibody). The Core device (with substrate tethered to streptavidin) is activated by incubating this lysate-antibody solution in the device for 2 days at 4°C and then removing it.

00140 Tumor specific T-cells are quantified by passing MARTI TCR+Jurkat cells (or wild type control Jurkat cells) over the activated device. T-cells are counted by bright- phase microscopy and by immunofluorescence.

00141 The results show that a majority of the TCR+Jurkat cells bind the device activated with MHC / MARTI antigen in amounts readily distinguishable from negative controls using isotype antibody instead of the anti-MHC antibody, or using MHC / tumor peptide antigen fraction from MART 1 negative cancer cells, or using non-transduced Jurkat cells

Example 7 Demonstration of Utility Using Primary Human T Cells

00142 This study used a human melanoma cell line SK-MEL-5 which expresses MART-1 peptides on HLA-A^*02:01 and a commercially available purified form of a MART-1 peptide (ELAGIGILTV) bound to HLA-A^*02:01 (purified-pMHC; MBL international Corp., MA, USA). Test T-cells (positive controls) used were commercially available primary CD8⁺ T-cells (Astrate Biologies, WA, USA) expressing T cell receptor (TCR) that recognizes the MARTI peptide ELAGIGILTV. The anti-MART-1 T cells were programmed using naturally processed native MART-1. The proportion of the anti- MART-1 T cells was >98%. Non-transformed Jurkat cells are used as negative controls.

00143 Reagents used included monoclonal antibodies against HLA-A2 (clone BB7.2), monoclonal antibodies against MARTI protein (clones A103 and M2-2C10), and monoclonal antibodies against human MHC (clone W6/32). The anti-HLA-A2 and anti- MART 1 antibodies were useful for immunofluorescence (e.g. on cell monolayers) or flow cytometry (performed on cell suspensions). The anti-human MHC antibody was used to purify peptide-MHC complexes and was used in our assay.

00144 The MHC / tumor peptide antigen fraction was prepared from cultured test cancer cells grown to confluence on monolayers. Cells were removed following mild trypsinization, treated with lysis buffer containing protease inhibitors, collected, and centrifuged at 500xg for 10 min. Supernatants were centrifuge at 100k xg and the pellet was resuspended in suspension buffer.

00145 Next, the core device was made according to Example 1 ; that is, it is coated in streptavidin. Cancer cell lysate or purified form of pMHC were incubated with biotinylated anti-MHC antibody (or control isotype antibody). The Core device (with substrate tethered to streptavidin) was activated by incubating this lysate-antibody or purified-pMHC-antibody solution in the device for 2 days at 4°C and then removing it.

00146 Next, the T-cells were labelled with a green fluorescent dye. The tumor specific T-cells were quantified by passing anti-MART 1 T cells (or wild type control Jurkat cells) over the activated device. T-cells were counted by immunofluorescence microscopy.

00147 As shown in Figure 7, anti-MART-1 T-cells flowing through the device were captured by time lapse recordings (left panel), demonstrating significantly enhanced capture of the T-cells in a device activated with purified-pMHC-antibody solution. In contrast, the numbers of captured anti-MART-1 T-cells in a negative control device activated with a purified-pMHC-isotype control antibody were substantially reduced (right panel). The T-cells are colored green, white arrows indicate lymphocytes flowing freely, yellow circles identify lymphocytes that have adhered to membrane. The numbers of captured wild type Jurkat cells in a device activated with purified-pMHC- antibody solution was also substantially low.

00148 . A majority of the anti-MART-1 T-cells were bound to the device activated with MHC / MARTI antigen from SKMEL-5 lysate) in amounts readily distinguishable from negative controls using isotype antibody instead of the anti-MHC antibody, or using wild type Jurkat cells.

00149 These data demonstrate that tumor reactive primary human T cells can be captured in the device and the naturally expressed peptide-MHC extracted from human cells can be used to activate the device. It should also be noted that this study validates the present invention because the model closely recapitulates claimed uses under the various clinical embodiments, We demonstrated here that it should now be possible to quantify tumor-specific leukocytes by applying a preparation of peptide- MHC complexes from a patient’s tumor to the core device and then isolating the tumor- specific leukocytes by passing peripheral blood over the device and binding them to the device (and then quantifying the retained leukocytes).

Example 8 Proof of Concept in Humans

00150 Twenty patients with newly diagnosed metastatic melanoma receiving front line checkpoint inhibitor therapy are enrolled in this study. A tumor sample is obtained from the initial confirmatory biopsy. Peripheral blood is obtained prior to the start of immune checkpoint blockade therapy (Day 0) and the sample is run through a device produced according to Example 1 - Example 3 to obtain an initial assessment of lymphocytes that are capable of specific recognition of and binding to tumor peptide antigens (i.e. Immunocompetence assessment).

00151 Repeat blood samples are obtained at Day 30 and at the time of the first cross-sectional imaging while on therapy (typically Day 90). In the event that toxicity develops or treatment cessation occurs prior to that time, a repeat Immunocompetence assessment is performed at the time of the first cross sectional imaging after treatment initiation. Results from Day 0, Day 30, and Day 90 are compared and correlated with imaging results and provider reported response rates.

Example 9 Correlation of Readout with Clinical Course

00152 The clinical course of the cancer in the eight patients from Example 8 is followed with time. It is discovered that patients that responded to immune checkpoint blockade therapy demonstrated an increase in Immunocompetence as measured by the present invention. In contrast, patients that had negligible or no response did not demonstrate an increase in Immunocompetence.

Claims

CLAIMS What is claimed is:

1. A device comprising: a vessel having a lumen; a fluid a substrate; an MHC binding agent; a peptide-MHC complex from a first subject; and a leukocyte, wherein: the substrate is in the lumen of the vessel; the MHC binding agent is attached to the substrate; the peptide-MHC complex is attached to the MHC binding agent; and the leukocyte is attached to the peptide-MHC complex.

2. The device of Claim 1 wherein the leukocyte is from the first subject.

3. The device of Claim 1 wherein the leukocyte is from a second subject.

4. The device of Claim 2 wherein the subject has a cancer and the peptide-MHC complexes is derived from the cancer.

5. The device of Claims 3 wherein the peptide-MHC complexes is derived from a non-cancerous organ.

6. The device of Claim 4 wherein the MHC binding agent is an anti-MHC antibody.

7. The device of Claim 6 further comprising a tether wherein the tether comprises a proximal end and a distal end and wherein the tether is attached to the substrate through the proximal end and the MHC binding agent is attached to the tether through the distal end.

8. The device of Claim 7 wherein the distal end of the tether comprises avidin or streptavidin and wherein the MHC binding agent is attached to biotin and the biotin is attached to the avidin or streptavidin, thereby attaching the MHC binding agent to the substrate.

9. The device of Claim 7 wherein the distal end of the tether comprises protein A or protein G and wherein the MHC binding agent is attached to the protein A or protein G, thereby attaching the MHC binding agent to the substrate.

10. The device of Claim 8 wherein the leukocyte is a lymphocyte.

1 1. The device of Claim 10 wherein the lymphocyte is a T cell.

12. The device of Claim 1 1 wherein the T cell is a PD-1 positive cytotoxic T cell.

13. The device of Claim 1 1 wherein the T cell comprises a T cell receptor and wherein the T cell is bound to the peptide-MHC complex through the T cell receptor.

14. The device of Claim 13 wherein the substrate is planar, spherical, or quasi spherical.

15. The device of Claim 14 wherein the tether is of a length greater than 10 nm.

16. The device of Claim 15 wherein the MHC binding agent is present at a density of between 1x10² and1x10⁶ molecules per mm² of substrate.

17. The device of Claim 16 wherein the substrate is glass.

18. The device of Claim 17 wherein the tether comprises a conjugate having the formula

( OH ) 2 0 0

X-0-Si-R₅-NH-C-R₆-C-NH-Q where X is a hydroxyl bearing Solid phase material; Rs is (CH2)(NH(CH)), where n is from 2 to 8, m is from 2 to 8, and p is from 0 to 3; R is selected from the group consisting of alkyl, cyclic alkyl, aromatic and heterocyclic groups containing 0 to 8 hydroxyl, hydroxycarbonyl, or aminocarbonyl groups, and Q is any molecule that contains a free primary or secondary amine group, wherein X is selected from the group of nitrocellulose, cellulose, glass fibers and porous glass beads.

19. The device of Claim 18 wherein the vessel comprises micro-channels.

20. The device of Claim 19 wherein the vessel has a first port and a second port wherein said first and second ports are configured to allow fluid to enter and exit the lumen of the vessel.

21. The device of Claim 20 wherein the vessel is a closed vessel but for the first port and second port.

22. The device of Claim 21 further comprising a pump configured to pump the fluid in to the first port through the lumen of the vessel and out the second port.

23. The device of Claim 22 wherein the pump is configured to create a sheer force of between 0.0001 dynes/cm² and 100 dynes/cm².

24. A method of collecting tissue-specific leukocytes from a subject comprising the steps of: a. affixing an anti-MHC antibody to a substrate wherein the substrate is located in the lumen of a vessel; b. obtaining a biopsy from a tissue of a first subject; c. processing the biopsy to obtain a preparation comprising soluble peptide- MHC complexes; d. incubating the preparation in the lumen of the vessel for a time sufficient for the peptide-MHC complexes to bind to the anti-MHC antibody; e. washing the lumen of the vessel to remove components of the preparation other than the peptide-MHC complexes; f. obtaining a leukocyte sample from the first subject or a second subject; g. incubating the leukocyte sample in the lumen of the vessel for a time sufficient for tissue-specific leukocytes to bind to the peptide-MHC complexes; and h. washing the lumen of the vessel to remove components of the leukocyte

sample other than the tissue-specific leukocytes, wherein the tissue-specific leukocytes are optionally cancer-specific leukocytes.

25. The method of Claim 24 further comprising quantifying the leukocytes bound to the vessel through the peptide-MHC complexes.

26. The method of Claim 25 wherein the quantification is performed by

photomicroscopy or immunofluorescence,

27. The method of any one of Claims 24 - 26 wherein the bound leukocytes are released from the vessel and collected.

28. The method of Claim 27 wherein the release is performed by shear force or proteolytic cleavage.

29. The method of Claim 27 wherein the released leukocytes are quantified.

30. A method of treating a subject with cancer comprising quantifying

Immunocompetence of the subject a first time by a device of any one of Claims 1 - 23, treating the subject with an immunotherapeutic agent, quantifying

Immunocompetence a second time, and continuing the immunotherapeutic agent treatment if Immunocompetence had increased at the second quantifying time compared to the first quantifying time and ceasing the immunotherapeutic agent treatment if there was no increase in the Immunocompetence at the second quantifying time.

31. The method of Claim 30 wherein the immunotherapeutic agent is a checkpoint therapeutic agent.

32. The method of Claim 30 wherein if there was no increase in the

Immunocompetence at the second quantifying time, treatment with a second immunotherapeutic agent is performed.

33. The method of Claim 31 wherein if there was no increase in the Immunocompetence at the second quantifying time, treatment with a second immunotherapeutic agent is performed.

34. A method of organ transplant comprising testing for Immunocompetence of a subject in need of a transplant against a peptide-MHC complex from a donor organ by a device of any one of Claims 1 - 23 and then transplanting the donor organ if no Immunocompetence is demonstrated and not transplanting the donor organ if Immunocompetence is demonstrated.

35. A kit comprising a device and instructions, wherein the device comprises a vessel having a lumen, a substrate, and an MHC binding agent; wherein the substrate is in the lumen of the vessel and the MHC binding agent is attached to the substrate; and wherein the directions for use comprise directions for one or more of:

(i) preparing the peptide-MHC complex; (ii) attaching the peptide-MHC complex to the MHC binding agent; (iii) preparing a leukocytes fraction; (iv) attaching leukocytes from the leukocyte fraction to the peptide-MHC complex; or (v) quantifying the leukocytes attached to the device.

36. The device of any one of Claims 1 - 23, wherein the leukocyte is attached to the peptide-MHC complex via protein-protein interaction; the peptide-MHC complex is attached to the MHC binding agent via protein- protein interaction; the MHC binding agent is attached to the substrate via covalent bonds; and the protein-protein interactions include one or more of electrostatic, hydrogen, hydrophobic, and Van der Waals; and optionally the MHC binding agent and/or its attachment to the substrate is man -made or synthesized.

37. A device comprising: a vessel having a lumen; a fluid a substrate; an MHC binding agent; a peptide-MHC complex from a first subject; and a leukocyte, wherein: the substrate is in the lumen of the vessel; the MHC binding agent is attached to the substrate; the peptide-MHC complex is attached to the MHC binding agent; the leukocyte is attached to the peptide-MHC complex. the leukocyte is from the first subject.; the subject has a cancer and the peptide-MHC complexes is derived from the cancer. the MHC binding agent is an anti-MHC antibody; the device further comprises a tether wherein the tether comprises a proximal end and a distal end and wherein the tether is attached to the substrate through the proximal end and the MHC binding agent is attached to the tether through the distal end; the distal end of the tether comprises avidin or streptavidin and wherein the MHC binding agent is attached to biotin and the biotin is attached to the avidin or streptavidin, thereby attaching the MHC binding agent to the substrate; the leukocyte is a T cell; the T cell comprises a T cell receptor and wherein the T cell is bound to the peptide-MHC complex through the T cell receptor; the tether is of a length greater than 10 nm; the vessel comprises micro-channels; the vessel has a first port and a second port wherein said first and second ports are configured to allow fluid to enter and exit the lumen of the vessel; the vessel is a closed vessel but for the first port and second port; the device further comprises a pump configured to pump the fluid in to the first port through the lumen of the vessel and out the second port; and the pump is configured to create a sheer force of between 0.0001 dynes/cm² and 100 dynes/cm².