Compositions and methods for negative selection of naive t cells and b cells with single antibodies

Abstract

Disclosed are compositions and methods for isolating naive, untouched target cells of interest.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application 63 / 031,184, filed May 28, 2020, the entire disclosure of which is incorporated herein by reference as if fully described. Technical Field

[0003] This invention relates to the field of immunology and to the magnetic separation and isolation of uncontacted immune cells, such as T cells, B cells, and NK cells, from biological fluids. Specifically, compositions and methods are provided for the efficient separation of unwanted cell types from biological samples, leaving the target cell types in an uncontacted, immature state, which can then be isolated and used for various therapeutic applications. Background Technology

[0004] Several publications and patent documents are cited throughout this specification to describe the prior art in the field to which this invention pertains. Each of these citations is incorporated herein by reference as if fully described.

[0005] Immunotherapy, which involves the selection, activation, expansion, and transfection of T lymphocytes (T cells) to produce chimeric antigen receptor (CAR) T cells, is one of the most important medical advances of this generation. FDA-approved products [for large B-cell lymphoma (Kymriah–Novartis) and for non-Hodgkin lymphoma (Yescarta–Gilead)] have been shown to cure certain leukemias and lymphomas. Many other applications of CAR T cells are being explored, and numerous clinical trials are underway globally (Kansagra AJ et al., Clinical Utilization of Chimeric Antigen Receptor TCells in B Cell Acute Lymphoblastic Leukemia: An Expert Opinion from the European Society for Blood and Marrow Transplantation and the American Society for Blood and Marrow Transplantation. Biol Blood Marrow Transplant. 25:e76-85, 2019; Seif M. et al., CAR T Cells Beyond Cancer: Hope for Immunomodulatory Therapy of Infectious Diseases. Front Immunol. 10:2711, 2019). As illustrated in a recent review, very significant efforts are being made to apply such therapies to solid tumors (Mohanty R. et al., CART cell therapy: A new era for cancer treatment. Oncol Rep. 42:2183-2195, 2019).

[0006] The manufacturing process of CAR-T cells (live drug) begins with the collection of peripheral blood mononuclear cells (PBMCs) from a patient or donor via apheresis. In the case of allogeneic CAR T cells, these cells can be (i) directly stimulated to activate and expand, thus allowing a sufficient number of cells to be processed in subsequent steps to produce a sufficient product, or (ii) T cells or subsets, such as CD4+, can be used. + and CD8 + T cell selection. Growing evidence suggests that cell selection as a starting step in manufacturing yields a better product. Furthermore, there is compelling evidence that CD4… + and CD8 +The initial T-cell ratio is important for providing more durable therapy (Turtle CJ., et al., CD19 CAR-T cells of defined CD4). + :CD8 + composition inadult B cell ALL patients. J Clin Invest. 126:2123-38, 2016).

[0007] Several T-cell selection methods have been used to produce CAR T cells. While there are many good biological reasons to start with “untouched” T cells, most processes involve positive selection using a targeting mAb that is specific to the CD3 receptor on the T cell, or on the CD4 receptor. + or CD8 + Suitable mAbs for positive selection of T cells. These mAbs can be used with common capture instruments, conjugated to separation carriers (such as magnetic nanoparticles, bubbly microbubbles, or other solid supports), or fluorescently labeled for selection of labeled cells by flow cytometry. None of these methods generate uncontaminated T cells. Furthermore, when particles are used in separation methods, they must be removed before proceeding with subsequent manufacturing steps. Failure to remove particles requires explicit justification that their presence on the starting cells will not have adverse effects—a tedious and costly task.

[0008] Positive selection methods for T cells and T cell subsets have certain advantages. For example, since most negative selection protocols require the removal of all non-T cell species from PBMCs, available negative separation systems typically use a mixture of monoclonal antibodies (mAbs) containing up to nine mAbs (minimum seven). These antibodies bind to specific surface receptors to remove non-target cells such as CD14-monocytes, CD15-granulocytes, CD16-NK cells and granulocytes, CD19-B cells, CD34-stem cells, CD36-monocytes / macrophages / platelets, CD56-NK cells, CD123-cells of the bone marrow lineage, and some B cells (when T cells are negatively selected) and CD235a-RBCs.

[0009] Please remember that, according to FDA guidance, any entity that comes into contact with precursor cells for therapeutic applications (in this case, a mAb) must have the same quality as a therapeutic mAb used in humans. Therefore, the start-up cost of using a large number of mAbs intended for in vivo use to create a negative selection system places a costly burden on healthcare systems and patients who need such therapeutic products. The total cost associated with performing such selection by piecing together commercially available positive selection kits to remove unwanted cells is approximately $20,000 per Leukopak. Adding these costs to current CAR T production costs is unacceptable. Summary of the Invention

[0010] According to the present invention, a method for isolating Fc receptor-negative target cell fractions from a peripheral blood mononuclear cell (PBMC) formulation is disclosed. In one embodiment, a PBMC formulation substantially free of endogenous or added IgG is provided. A single immunologically active trapping agent is introduced that binds simultaneously to epitopes on both Fc receptor-carrying cells and B cells, wherein the trapping agent is operatively linked to a ferrofluid comprising magnetically responsive particles and forms a magnetic cluster of Fc receptor-carrying cells, including B cells, monocytes, granulocytes, and platelets; the magnetic clusters of Fc receptor-carrying cells and B cells are then isolated from the formulation in a magnetic separator, and the target cell fraction in a substantially immature, uncontacted state is recovered.

[0011] In another embodiment for the isolation of uncontacted Fc receptor-negative target cells, a single immunoactive trapping agent that binds simultaneously to both Fc receptor-carrying cells and epitopes on B cells is introduced into the PBMC formulation, wherein the trapping agent is operatively linked to a first member of a specific binding pair. The formulation is then contacted with a ferrofluid containing magnetically responsive particles operatively linked to a second binding member, under conditions where a specific binding pair has formed between the first and second binding pair members, thereby forming magnetic clusters of Fc receptor-carrying cells selected from B cells, monocytes, granulocytes, and platelets. The magnetic clusters of cells are then isolated from the formulation in a magnetic separator, and a fraction of target cells in a substantially immature state is recovered. B cell epitopes used in the methods disclosed herein include, but are not limited to, CD19, CD20, IgG, and CD32.

[0012] In another embodiment of the method for isolating Fc receptor-negative target cells in an uncontaminated state, anti-human IgG and a capture agent comprising a first member of a specific binding pair, which is Fab or F(ab)' μCells, each of the IgG and Fab or F(ab)'2 having affinity for B cell epitopes, are introduced into a PBBC ​​formulation having reduced endogenous IgG levels. Under conditions where specific binding pairs are formed between the first and second binding pair members, the PMBC formulation is contacted with a ferrofluid containing magnetically responsive particles operatively linked to a second binding member, thereby forming magnetic clusters of cells carrying Fc receptors, including IgG-bound B cells, monocytes, granulocytes, and platelets. The magnetic clusters of cells are then separated from the formulation in a magnetic separator, and a fraction of target cells in a substantially immature state is recovered.

[0013] In some preferred embodiments, the target cells are CD3 cells. + T cells. In other embodiments where antibody specificity is altered, the aforementioned clustering characteristics can be used to isolate B cells. CD34 can be isolated by providing appropriate binding pair members. + stem cells, CD4 + CD8 + and NK cells.

[0014] In cases where untouched T cells are to be recovered, the capture agent used in the above methods comprises, for example, a mouse or human monoclonal IgG antibody containing an Fc region that binds to human FcγR on non-target cells, the antibody having binding affinity for epitopes on B cells. In some preferred embodiments, the antibody is IgG1. In other methods, IgG is added together with an immunologically active antibody fragment (e.g., Fab), wherein the fragment is operatively linked to the first member of a specific binding pair. In embodiments where non-target cells carrying FcγR are to be removed, such cells include monocytes, granulocytes, macrophages, dendritic cells, and NK cells.

[0015] While this article exemplifies the use of biotin and streptavidin, other useful binding pairs include, but are not limited to, receptor-ligand, agonist-antagonist, lectin-carbohydrate, avidin-biotin, biotin analog-avidin, desulfobiotin-streptavidin, desulfobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

[0016] It also provides a method for isolating immature CD4 from peripheral blood mononuclear cell (PBMC) preparations. + or CD8 + T cell methods. In order to isolate immature CD4 cells... + In the cellular implementation scheme, a first immunomodulatory trap binds simultaneously to both epitopes on cells carrying the Fc receptor and B cells, as well as to CD8. +A second immune-active trapping agent that binds to T cells is introduced into the PBMC formulation. In this case, each of the first and second immune-active trapping agents is operatively linked to magnetically responsive particles present in a ferrofluid, which forms CD8 + Magnetic clusters of T cells, B cells, and cells carrying Fc receptors. The magnetic clusters of cells are separated from the formulation in a magnetic separator, and CD4+ in a substantially immature state is recovered. + T cells. When CD8 needs to be isolated... + In the case of T cells, anti-CD4 antibody can be used instead of anti-CD8 antibody.

[0017] This invention also provides a method for isolating immature NK cells from a peripheral blood mononuclear cell (PBMC) formulation under conditions suitable for high-affinity Fc receptor binding. In this embodiment, a first immunologically active trapping agent binds simultaneously to both Fc receptor-carrying cells and epitopes on B cells, and CD3... + Under conditions that promote high-affinity FcR binding, each of the first and second immune-active trapping agents is operatively linked to magnetically responsive particles present in a ferrofluid and forms Fc receptor-carrying cells, B cells, and CD3+ cells. + Magnetic clusters of cells. The magnetic clusters are then separated from the formulation in a magnetic separator, and the immature NK cells in a substantially immature state are recovered.

[0018] On the other hand, it provides a method for isolating immature CD34 from peripheral blood mononuclear cell (PBMC) preparations. + A stem cell approach. An exemplary method involves introducing into a PBMC formulation: an immunologically active trapping agent that simultaneously binds to epitopes on both Fc receptor-carrying cells and T cells, the immunologically active trapping agent being operatively linked to magnetically responsive particles present in a ferrofluid; the Fc receptor-carrying cells and T cells forming magnetic clusters, the magnetic clusters being segregated from the formulation in a magnetic separator, thereby allowing the recovery of CD34 in a substantially immature, untouched state. + Stem cells. In a preferred embodiment of the method, PBMCs are isolated and treated with G-CSF to induce hematopoietic stem cells to migrate from the bone marrow to the donor in the peripheral blood. Attached Figure Description

[0019] Figure 1This is a schematic diagram of the binding partners for negative selection of naive, untouched CD3 cells, as described in the examples below. Anti-CD32 mAb binds to CD32-expressing cells via Fab complementary site-CD32 epitope interaction and to other FcγRs via Fc-FcγR interaction. CD19 and CD20 are B cell-specific receptors; anti-CD19 and anti-CD20 bind to B cells via specific CD19 and CD20 epitopes, respectively, and also to other cells via Fc-FcγR interaction. sIg is also a B cell surface-specific receptor; anti-IgG mAb binds to B cells via sIg epitopes and to other cells via Fc-FcγR interaction. Furthermore, anti-IgG can also be used to bind plasma-free Ig labeled with FcγR-expressing cells. When using anti-IgG antibodies, the antibody can be in full-length IgG form or in Fab / F(ab') form with affinity for plasma-free Ig to label FcγRC.

[0020] Figure 2 Histograms are shown after cell labeling and isolation with MAH-Ig-FF and subsequent flow cytometry analysis. Cells were gated on CD45-FITC, and CD3, CD11b, and CD19 positive cells were measured after cell selection. Detailed Implementation

[0021] A growing body of clinical data suggests that adoptive transfer of genetically engineered T cells, such as chimeric antigen receptor (CAR) T cell-mediated immunotherapy, is effective in treating cancer and autoimmune diseases. To date, the production of immune cells has primarily relied on T cell positive selection, based on mAb labeling of specific receptors expressed on these cells. This strategy can lead to alterations in gene expression because the positive selection process requires contacting cells with reagents that can induce unwanted or premature cell activation, mediating activation-induced cell death after isolation. Furthermore, there are anecdotal reports that positive cell selection can lead to antibody-dependent cytotoxicity shortly after transfer to the recipient. These drawbacks are likely to adversely affect the potency and long-term persistence of the immune cells used. Therefore, an alternative strategy is to utilize desired cell types, such as naive T, B, NK, or CD34 cells. + Simplified negative selection of stem cells will be of great value and will reduce unwanted activation of target cells.

[0022] The leukocyte composition of PBMCs / Leukopaks is as follows: T cells (45-60%), B cells (5-15%), monocytes (10-30%), granulocytes (0.6-10%), and NK cells (5-10%). To date, there is no negative selection protocol for preparing CAR T cells for clinical applications. However, for PBMCs / Leukopaks separated using magnetic separation, several negative selection options exist at routine research levels. For example, in the Miltenyi Biotech (Bergisch Gladbach, Germany) pan-T cell selection kit (#130-096-535), the antibody mixture contains nine antibodies (CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a) to remove non-T cells from PBMCs; similarly, in Dynabead-based CD3 negative selection products, seven antibodies (CD14, CD16, CD19, CD36, CD56, CD123, and CD235a) are involved, with comparable purity (~95% and above) (Dynabeads). TM Untouched TM Human T-cell kit, #11344D). One kit uses three antibodies, CD14, CD19, and CD56, to negatively enrich CD3. + Reports of T cells yielded similar results; however, Ficoll purification was required to remove granulocytes before isolation (Janssen W. et al., A simple large-scale method for T cell enrichment by negative selection in preparation for viral transduction. Cytotherapy 19:S38,2017).

[0023] In previous studies on CD3-positive T cell selection (Liberti PA, Riter DW, Khristov TR. A novel low-cost high-yield clinical scale cell separator. Cell Gene TherapyInsight. 4:581-600, 2018), an indirect method of magnetically labeled T cells was used to achieve positive immunomagnetic separation. In these studies, PBMCs were first incubated with anti-CD3 mAbs and then magnetically labeled with a common capture version of our proprietary 135–165 nm highly magnetic nanoparticles (also known as ferrofluids or FF) (Liberti et al., US Patent Nos. 5,698,271; 6,120,856). Preliminary studies began using a common capture agent, namely a rat anti-mouse Fc(RAM) mAb conjugated with FF. This system consistently produced cells with excellent CD3-positive T cell selection. + T-cell products with a cell yield (>75%) were repeatedly contaminated with varying levels of monocytes (monocytes comprised 10-95% of the PBMC formulation). Significant attempts to inhibit monocyte binding were made based on the hypothesis of RAM-FF and FcR interaction, including pre-incubation of PBMCs with heat-accumulated human IgG (HAIG) and FcR blocking antibodies. Monocyte binding could not be inhibited regardless of HAIG levels, while the Fc blocking antibody was effective. However, the cost of using such an inhibitor has hindered the manufacture of commercially viable isolation kits.

[0024] Analysis of numerous different RAM-FF formulations used in early positive-selective T-cell studies revealed a strong correlation between the RAM level used for FF conjugation and monocyte contamination (capture). The analysis showed that at certain levels of RAM conjugation with FF, these nanoparticles bind affinitylessly to cells carrying FcγR (FcγRC), demonstrating the important role of RAM Fc proximity in its coupling with FF and other solid surfaces or supports. The role of RAM Fc proximity on solid supports such as our FF has been experimentally demonstrated in our laboratory by effectively saturating the FF surface with RAM (4000–7000 IgG / particle) and comparing its affinity for monocytes with that of low-level conjugation (500–1000 IgG / particle) and other conjugated non-immune proteins. FF prepared with high levels of RAM can completely remove monocytes from aliquots of ablation products. Furthermore, this removal can be achieved using the same concentration of FF used in mAb-targeted cell separation. Note that RAM is an IgG1 subtype. Furthermore, when PBMCs are incubated with FF conjugated with bovine or human serum albumin (BSA, HSA), monocytes cannot be magnetically removed regardless of the conjugation level.

[0025] To determine whether monocytes produce magnetically responsive cells through RAM-FF uptake rather than RAMFc:FcγR binding interactions, experiments were performed at 0°C and yielded the same results as those performed at room temperature. When experiments were conducted with lower levels of RAM conjugated to FF, lower levels of monocytes were recovered. All these findings suggest that the RAM-FF:monocyte interaction is largely binding in nature and requires close proximity of the RAM Fc region or is enhanced by close proximity of the RAM Fc region.

[0026] These findings led us to investigate the following hypothesis: when the conjugation level of a single antibody to a solid support is sufficiently high, it should be possible to effectively target CD3 with a single antibody against an IgG subclass. +T-cell negative selection is employed because, at such levels, the Fc region of such antibodies would be sufficiently close to act as an affinity conjugate for FcγR, while simultaneously, the antibody's specificity would be available for binding to at least one other cell specificity, namely B cells. Thus, the proximity of the Fc region on a solid support enhances affinity through multivalent attachment to neighboring FcγRs. To further investigate T-cell negative selection using a single mAb, mouse anti-human IgG antibodies of the IgG1 isotype were conjugated to FF at high levels (4000–7000 mAb / particle). The working hypothesis of this experiment is that the Fab variable domain binds to B cells because B cells express surface IgG, and that the close proximity of the antibody's Fc region to the surface binds to FcγR expressed on monocytes, macrophages, dendritic cells (DCs), granulocytes, natural killer (NK) cells, and B cells because B cells express FcγRIIB.

[0027] Experiments conducted using numerous permutations and controls have validated the hypothesis and demonstrated that, through this strategy, we can repeatedly isolate immature CD3 with 95% purity or higher. + T cells. For some experiments, PBMC formulations are essentially free of endogenous IgG because they are washed with three centrifugal cycles to avoid endogenous IgG, which would obviously react with anti-human IgG antibodies. When human IgG is added to such PBMC formulations at levels greater than about 4 to 10 μg / mL, T cell recovery decreases while B cell contamination increases. Given the use of anti-human IgG to label B cells in those experiments, we infer that endogenous IgG would effectively neutralize the cell-labeling ability of anti-human IgG antibodies. In embodiments, when using Fab or Fab-like fragments linked by disulfide, such as F(ab')2, low levels of endogenous IgG would aid in selection, i.e., biotinylated Fab or F(ab')2 would bind to B cell surface IgG to label B cells, while Fab / F(ab')2 could also bind to free plasma IgG to form F(ab')2-IgG complexes and label FcγRC for removal ( Figure 1 To make a sample “substantially free of IgG”, at least three washes and resuspensions are required. When using Fab / F(ab')2, cells are washed only once or twice, thus preserving residual plasma IgG in the sample.

[0028] Since anti-human IgG antibodies can bind to endogenous IgG in cell products as well as Ig that binds to FcγRC, to simplify the model, anti-CD19 mAb was conjugated at a high level (4000-7000 mAb / particle) to FF and used for negative selection of PBMCs that do not lack endogenous IgG. This experiment also resulted in the production of highly pure (>92%) naive T cells. Based on this conceptually simple experiment and a comparison with the anti-human Ig experiment described above, we hypothesize that one component reacts specifically with B cells, and the other component reacts via Fc and FcγRC, which are most likely to aggregate, is sufficient to produce naive T cells.

[0029] Using a single antibody to enrich target cells significantly simplifies the cell labeling process, dramatically reduces the cost of traditional protocols involving numerous antibodies, and results in a surprisingly rapid process. The appeal of this concept, supported by the experimental data presented below, will greatly accelerate its translation into clinically relevant products. The same strategy can also be used to simplify CD4... + or CD8 + Negative selection of T cells, B cells, and NK cells, as described below, can be accomplished by adding an additional mAb to the system.

[0030] As described above, our initial selection of T cells from PBMCs was performed using an indirect magnetic labeling method employing the following steps: (i) incubation with anti-CD3 mAb, (ii) removal of unbound mAbs, and (iii) incubation with RAM-FF. Mononuclear cell contamination in these selection protocols was entirely unacceptable. If, alternatively, for similar positive selection experiments, T cells were labeled with biotinylated anti-CD3 mAb, followed by removal of unbound mAbs, incubation with SA-FF, and magnetic separation, a significant improvement in mononuclear cell contamination was observed, but it still remained at unacceptable levels. This problem was addressed by first incubating PBMCs, which were essentially devoid of endogenous IgG, with only 1.0 mg / mL of human IgG, followed by incubation with a biotinylated anti-CD3 antibody to remove unbound mAbs, and then magnetic labeling with SA-FF. These experiments resulted in T cell preparations that were essentially free of mononuclear cell contamination. These results suggest that the interaction of biotinylated anti-CD3 mAb with FcRs, particularly high-affinity receptors, may play a role in mononuclear cell contamination without the addition of IgG. It is important to remember that 1 mg / mL of human IgG was needed in the above experiment to prevent mononuclear cell contamination, which is approximately 1000 to 1 relative to the labeled mAb.

[0031] We then considered the possibility that biotin-antiCD3 mAb incubated with PBMCs in the absence of other Ig might exchange with endogenous IgG bound to FcγR, or the existence of a sufficient number of “empty” FcRs so that the added labeled mAb could occupy such sites, thus leading to the possibility of targeted anchors for labeling FcγRCs. Due to the different K values ​​of the binding reactions of FcγRs to monomeric IgG... d The range is 10 -6 Up to 10 -9 M,IgG should bind with sufficient energy to be used as a labeling agent. Furthermore, due to the multivalent nature of our SA-FF, there is an opportunity to enhance the binding energy between FcγRC and SA-FF through multivalent attachment of biotinylated mAbs to FcγRC, making the Fc–FcγR interaction of the mAb a facilitating factor.

[0032] These findings are particularly relevant to any negative isolation assay. A preferred approach is to retain the unbound labeled mAb in the reaction mixture before adding a common trapping agent. This provides an opportunity for such reagents (SA-FF in this case) to react with mAb-labeled B cells as well as any other biotin-carrying components. For example, in the case of using a biotin-anti-CD19 mAb, incubating the mAb with PBMCs can result in mAb-labeled B cells and mAb labeling of FcγR on FcγRC. Upon addition of a common trapping agent such as SA-FF, multiple reactions may occur, including the latter binding to mAb-labeled B cells, binding to any biotin-carrying FcγRC, and binding to unbound biotin-anti-CD19. Therefore, during magnetic labeling incubation, in addition to the labeling reaction with B cells, a new class of reactions is formed: a complex of SA-FF with the bound antibody. Based on RAM-FF data, as stated above, we predict that this complex will bind affinitylessly to FcγRC. Therefore, the use of B cell-specific mAbs (such as CD19, CD20, or IgG) in indirect selection protocols can involve many complex reactions in which common trapping agents can play a variety of advantageous roles. For example, the multivalent nature of SA on nanoparticles or surfaces can promote the labeling of FcRCs by biotin-antibodies binding to them, in which case the multivalent nature of SA-FF increases the binding strength to a level that can capture such FcRCs. The same multivalent nature of SA-FF and its reaction with unbound biotin-mAbs generate a class of cells that can react with unfilled FcRs, thus creating another mechanism for labeling FcR cells (FcRCs) for subsequent removal.

[0033] Based on our studies of positively selected T cells using SA-FF and the effect of added human IgG on reducing monocyte contamination, it appears that added IgG may counteract the binding of multivalent SA-FF to biotinylated mAbs that bind to FcγR, since these mAbs are expected to compete with FcγR when the concentration of added IgG is high enough, especially after the removal of unbound biotinylated anti-CD3 mAbs in those experiments.

[0034] To gain insight into the possible mechanisms involved in FcγRC removal, the following negative T-cell selection assay was performed: Biotinylated anti-CD19 mAb was incubated with PBMCs in the absence of IgG, followed by incubation with SA-FF and magnetic separation. T-cell purity >92% was obtained, with the T-cell fraction showing almost no monocyte contamination. As expected, the presence of IgG (0.125–1 mg / mL) did not significantly alter monocyte eradication. Furthermore, monocyte removal became more complete with increasing amounts of biotinylated anti-CD19 mAb (e.g., 2–4 μg / mL superior to <2 μg / mL), and CD3 purity was higher. Based on these results, it appears that biotin-mAbs, which are capable of binding to biotin-mAbs in a closed-packed manner, can affinityally bind to FcRCs.

[0035] These disclosures indicate that there are multiple ways to enable FcγR to participate in cell separation in a very active manner, thereby creating multiple pathways to isolate immature, untouched cells in a simple and reagent-saving manner.

[0036] definition:

[0037] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, each of the following terms shall have the meanings described below unless otherwise expressly specified herein. Further definitions are shown throughout the application.

[0038] The term "biological sample" includes, but is not limited to, cell-containing body fluids, peripheral blood, tissue homogenates, aspirates, and any other source of rare cells available from human subjects.

[0039] The term "determinant cluster" refers to a target cell portion that can be specifically bound by a biospecific ligand or biospecific reagent when used with any target cell described herein, and is involved in and responsible for the selective binding of the specifically binding substance, the presence of which is required for selective binding. Essentially, a determinant cluster is a molecular contact region on a target cell that is recognized by a receptor in a specific binding pair response.

[0040] As used herein, the term "specific binding pair" includes antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, biotin-streptavidin, nucleic acid (RNA or DNA) hybrid sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin, and virus-receptor interactions. Various other cluster-specific binding combinations are considered for use in carrying out the methods of this invention, as will be apparent to those skilled in the art. When the first member of a specific binding pair, such as an anti-CD3 mAb, binds to its second member, namely a CD3 epitope on T cells, such a reaction is called a "labeling reaction," and these T cells are considered to have been labeled with the mAb.

[0041] "Positive selection" refers to purification from a mixture of different attachments of the first member of a specific binding pair, whereby the first member selectively binds to the second member of a second binding pair present on a target cell type, thereby isolating the cells from the mixture. Various methods and approaches for positive selection, i.e., purification of target entities, using the second member of a specific binding pair are well known in the art.

[0042] "Negative selection" refers to the purification of a target cell type from a mixture of different cell types by attaching one or more first members of one or more specific binding pairs to each cell type in the mixture other than the target cell type. Specific binding pair reactions using the second member of the binding pair allow entities carrying the first member of the binding pair to be separated from the mixture, leaving the target entity. The methods and approaches used to perform such separations are well known in the art. The remaining portion of the mixture is called the negative fraction.

[0043] "A essentially immature or untouched state" refers to a subpopulation of cells that has not been contacted by any specific binding pair members.

[0044] "T cells" refers to CD3 cells. + Cells. By definition, CD3 + Negative selection of cells produces naive T cells that have not come into contact with any specific binding pair.

[0045] "Fc receptor-negative target cells" are target cells that express little or no Fc receptors.

[0046] In positive and negative selection, the cell types to be recovered or eliminated are typically contacted with one member of a specific binding pair, such as an epitope on those cells that have reacted with the corresponding antibody, or with a second member of the specific binding pair, which binds specifically to that epitope with high affinity. This high-affinity binding pair reaction is often referred to as a “labeling reaction.”

[0047] The term "antibody" includes, but is not limited to, glycoprotein immunoglobulins that specifically bind antigens. Typically, an antibody may comprise at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, or an antigen-binding molecule thereof. Each H chain contains a heavy chain variable region and a heavy chain constant region. The heavy chain constant region contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region and a light chain constant region. The variable regions of both the heavy and light chains contain binding domains that interact with the antigen. The portion of the antibody molecule formed by the interaction of the light and heavy chain variable regions (VL and VH) and their constant regions (CL and CH1) is called Fab or Fab fragment (antigen-binding portion). Papain digestion of the antibody results in the production of Fab fragments (two fragments per molecule) and crystalline portions called Fc (crystalline portions). The Fc fragment consists of heavy chain domains below the hinge region and is formed by the interaction of CH2 domains with each other, and similarly with CH3 domains. Pepsin digestion of antibodies cleaves the antibody molecule below the hinge region, leaving the disulfide bonds linking the heavy chain intact. This results in two Fab-like fragments linked by disulfides, referred to as (Fab')2. When those disulfides are reduced, a single antigen-binding fragment (Fab') is produced. The hinge region and CH2 domain of the heavy chain (the region just below and near the hinge region) contain the region of the antibody molecule that binds to the Fc receptor (FcR) on the cell. Because pepsin digestion of antibodies does not always result in uniform cleavage, some (Fab')2 formulations may contain FcR-binding sequences.

[0048] Antibodies or immunoglobulins (Ig) can be derived from any commonly known isotype, including but not limited to IgG, IgM, IgE, IgA, and secretory IgA. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG. lIgG2, IgG3, and IgG4 or mouse IgG1, IgG2, and IgG3. For example, the term "antibody" includes both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; recombinant antibodies; immunoglobulins, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-heavy chain pairs, intrabody antibodies, antibody fusions, heteroconjugate antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), affinity antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimics"), and any of the antigen-binding fragments described above. Antibodies as defined above can be obtained from any kind.

[0049] Cells expressing Fc receptors (FcRCs) differ from other hematopoietic cells in their ability to adhere to antibody-antigen complexes. FcRs bind to amino acid sequences primarily located in the CH2 domain of certain antibody subclasses. FcRs also bind to sequences on the antibody heavy chain located below or at the hinge, and close to the CH2 hinge region of certain antibody subclasses. FcγRs are a class of FcRs that specifically bind to certain subclasses of IgG antibodies. These transmembrane molecules recognize the Fc regions of various immunoglobulin (Ig) classes and subclasses. The terminology for different isotypes of FcRs and their corresponding CD nomenclature are as follows: for IgG (FcγRI / CD64, FcγRII / CD32, and FcγRIII / CD16), IgE (Fc∈RI), IgA (FcαRI / CD89), IgM (FcμR), and IgA / IgM (Fcα / μR). The phrase “basically free of FcR” means that cells do not adhere to surfaces carrying immune complexes, which are formed by Ig isotypes and subtypes known to bind to FcR.

[0050] "Isotype" refers to an antibody class or subclass (e.g., IgM or IgG1) encoded by a gene in the heavy chain constant region.

[0051] The term "detectable label" refers to any substance whose detection or measurement, directly or indirectly, by physical or chemical means, indicates the presence of target cells in a test sample. Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that can be detected directly or indirectly based on their light absorption, fluorescence, reflection, light scattering, phosphorescence, or luminescence properties; molecules or ions that can be detected by their radioactivity properties; and molecules or ions that can be detected by their nuclear magnetic resonance or paramagnetic properties. For example, included in the group of molecules that can be detected indirectly based on light absorption or fluorescence are various enzymes that cause appropriate substrate transformations, such as from non-light-absorbing molecules to light-absorbing molecules, or from non-fluorescent molecules to fluorescent molecules.

[0052] The phrase “substantially excluded” refers to the specificity of the binding reaction between a biospecific ligand (e.g., mAb) or a biospecific reagent (e.g., biotin and streptavidin) and its corresponding target determinant (e.g., cell receptor on the target cell). Biospecific ligands and reagents may exhibit specific binding activity to their target determinants, but may also show low levels of nonspecific binding to other sample components.

[0053] As used in this article, the term “enrichment” refers to the enrichment of target T cells and B cells from biological samples.

[0054] Preferred magnetic particles for carrying out the present invention are colloidal particles. Such particles are characterized by their submicron particle size, typically less than about 200 nanometers (nm) (0.20 micrometers), and their long-term stability in separating from solution under gravity. This size range, among many other advantages, makes them essentially invisible to analytical techniques commonly used in cell analysis. Particles in the 90-150 nm range with 70-90% magnetic material are considered for use in the present invention. Suitable magnetic particles consist of a crystalline core of a superparamagnetic material surrounded by molecules that bind (e.g., through physical absorption or covalent attachment) to the core and impart stable colloidal properties. The coating material should preferably be applied in an amount that effectively prevents non-specific interactions between biomolecules present in the sample and the magnetic core. Such biomolecules may include sialic acid residues, lectins, glycoproteins, and other membrane components on non-target cell surfaces. Furthermore, the material should contain as much magnetic material / nanoparticles as possible. The magnetic crystals containing the core are small enough that they do not contain intact magnetic domains. The nanoparticles are small enough that their Brownian energy exceeds their magnetic moment. Therefore, even in moderate magnetic fields, the north-south pole alignment and subsequent attraction / repulsion of these colloidal magnetic particles do not appear to occur, thus contributing to their solution stability. Finally, the magnetic particles should be separable in high magnetic gradient external field separators. This property facilitates sample handling and provides an economic advantage over more complex internal gradient columns loaded with ferromagnetic beads or steel wool. Magnetic particles possessing the aforementioned properties can be prepared by modifying the base materials described in U.S. Patent Nos. 4,795,698, 5,597,531, and 5,698,271. The preparation of these base materials is described below.

[0055] Effectively isolate immature, untouched target cells

[0056] This article describes a method for preparing immature cells, such as T cells, for use in genetic engineering methods and therapeutic approaches, such as adoptive cell therapy. Specifically, in some embodiments, the method uses or generates compositions containing multiple different cell populations or cell types, such as isolated CD4+. + and / or CD8 + T cell populations. In some embodiments, the method includes the step of isolating one or more cell populations. The cells used to perform the methods described herein are typically derived from samples from mammalian subjects, preferably human subjects.

[0057] We have discovered several methods for negative selection of multiple important cell populations using FcRs on FcRCs with a minimal number of specific antibodies. In the case of T cells, we found that a single antibody, preferably an IgG antibody, with its amino acid residues below and close to the hinge region, the CH2 domain, or the intact Fc can bind to FcγRCs. In the absence of substantially endogenous or added IgG besides the labeled antibody, the Fab moiety of such antibodies reacts with unique epitopes on B cells and is effective for labeling all cells in PBMC formulations except naive and memory T cells, while in other cases it reacts with IgG present. Combined with a range of known cell isolation techniques, such as specific binding-pair reactions using solid supports and flow cytometry-type cell sorting methods, our findings enable the purification of naive T cells with a single mAb or highly specific polyclonal antibodies. In other words, FcγRs expressed on monocytes, macrophages, dendritic cells (DCs), granulocytes, and NK cells, as well as the Fc moiety of antibodies interacting with FcγRs, can be creatively and advantageously used for cell isolation and purification of naive T cells. When this discovery is combined with the use of single mAbs targeting B cell epitopes (such as surface Ig, CD19, CD20, or CD32 on B cells), a simple, efficient, and extremely cost-effective method for preparing naive T cells is achieved. This is further enhanced by combining this method with a single mAb targeting CD4. + or CD8 + The cell's second mAb, immature CD4 + or CD8 + Cells are readily prepared. This invention has several embodiments.

[0058] In one embodiment for generating naive T cells, essentially labeling all cells in the PBMC except naive T cells for subsequent removal is achieved by attaching Fc fragments from an antibody class that binds to FcγR to a solid support and another entity capable of binding to B cells with sufficient binding capacity. In both cases, FcγR and B cells are removed from the suspension, respectively. This can be achieved using a single antibody with unique B cell specificity and a subclass that binds to FcγR. Notably, we have determined that the density of Fc on such surfaces affects the binding interaction with FcγR or with Ig bound to FcγR, and that the proximity of Fc on this surface must be taken into account. We hypothesize that these interactions are enhanced through multivalent binding interactions known in biological processes.

[0059] Several B-cell-specific determinants or epitopes exist that can be targeted for the aforementioned purposes, such as CD19, CD20, and surface Ig. The expression of these B-cell-specific epitopes is highly stable during B-cell development. Another suitable epitope is CD32, which is preferred among other B-cell-specific epitopes due to its widespread expression. CD32 is expressed on B cells and other WBCs, except for T cells. This widespread expression provides an efficient method for labeling all FcγRCs via strong Fab / CD32 epitope binding and Fc / FcγR binding. In this application, we describe several mAbs suitable for this task. Simply coupling any of these FcγR-reactive mAbs to a surface at an appropriate density produces a solid support that binds to both B cells and FcγRCs. With one of these mAbs coupled to a culture dish or similar container, researchers or diagnosticians can easily prepare naive T cells simply by removing unwanted cells. When such mAbs are coupled to nanoparticles (such as FF used in this paper), other magnetic particles, or nano / micron entities with levitation capabilities, larger-scale separation can be achieved using simple methods. Several methods exist for attaching mAbs directly to surfaces or coupling them via suitable joints. They can be coupled via joints, which can be advantageous, but as we have shown, direct coupling works well.

[0060] It should be understood that antibody fragments can be used in the above-described embodiments. Methods for producing Fab and Fc fragments have been known for nearly 50 years and are readily available, as have methods for preparing the CH2 domain, which is a major component of the IgG molecule that binds to FcγR. Therefore, the amino acid sequence of the Fc or CH2 fragment, or even the amino acid sequence just below and near the hinge region (Kiyoshi M. et al., Structural basis for binding of human IgG1 to its high-affinity human receptor FcγRI. Nature Communications, (2015), 6:6866), can be immobilized with an anti-B cell-specific entity to generate a strong and specific solid support for capturing and subsequently removing FcγRC and B cells. These fragments can be directly linked, but using adapters may be more efficient, as the latter approach generates more stereochemical binding opportunities.

[0061] In another embodiment for generating naive T cells with some of the advantages provided by the indirect labeling process and the benefits of a conventional capture agent, PBMCs are incubated with a mAb targeting a B-cell epitope (e.g., anti-CD19, anti-CD20, anti-human Ig, or anti-CD32). This is then incubated with the conventional capture agent on a suitable support carrying an anti-Fc against the species from which the labeled mAb originated. The requirement for this anti-Fc is that it must be a subtype capable of strongly interacting with FcγR known in the art. Although this embodiment requires two mAbs (a labeled mAb and a conventional capture mAb) for clinical use, the labeled mAb, i.e., the anti-B-cell mAb in molecular form, can be used effectively, while the second mAb on the conventional capture agent can be used in this manner for many other specific isolation processes.

[0062] To this end, we used monoclonal rat anti-mouse Fc (RAM) conjugated with FF at high density, and as previously mentioned, we also used SA bound to a solid support and biotinylated targeting mAbs.

[0063] It is noteworthy that in indirect approaches using RAM coupled to a solid support to label FcγRC, two classes may be involved in the labeling reaction: the RAM-solid support and the component bound to the targeting antibody, provided they belong to the appropriate subclass. Alternatively, different mechanisms may exist when using SA-conjugated solid supports or other specific binding pairs. In these cases, common traps are likely to become labelers of FcγRC when they bind to the targeting mAb, and also, possibly through their multivalent nature, when bound to biotin-labeled mAbs related to FcγR. Other specific binding pair reactions known in the art exist, such as DNP / anti-DNP, fluorescein / anti-fluorescein, biotin / anti-biotin, and arsenoic acid / anti-arsenoic acid, which can be used as alternatives to biotin / streptavidin; however, the strength of the biotin / SA binding pair is very close to that of existing covalent bonds. In this negative selection process, reversal labeling is not required. If a reversal reaction is needed for the removed cells in the future, binding pairs with lower binding affinity can be used, such as dethiobiotin and streptavidin or biotin-antibiotin binding pairs, which can dissociate from avidin or streptavidin. Other methods for breaking the bonds between mAb and biotin or between streptavidin and HSA can also be considered.

[0064] Because PBMCs can be divided into three subgroups or fractions—naive T cells, B cells, and FcγRC—any method that removes the latter two groups should result in the segregation of naive T cells. The above-described embodiment leverages the commonality of FcγR across many cell types and, by targeting such cells and combining them with an anti-B cell mAb coupled to a solid support, will generate naive T cells through appropriate treatment. Furthermore, the labeling and separation treatment steps can be performed indirectly using a biotinylated mAb or a conjugated fragment thereof with a common SA trapping agent. The SA can be coupled to any of the aforementioned nano / microparticles or can be attached to column packing materials such as agarose or fibers, as well as other materials known in the art (see, for example, Etchells and Peterson, U.S. Patent No. 5,215,926).

[0065] As mentioned above, PBMCs can be divided into three subgroups or grades: naive T cells, B cells, and FcγR cells, and an antibody specifically targeting B cell epitopes can effectively inhibit CD3. + T cells are isolated to high purity. In fact, B cells also express low-affinity FcγR and FcγRIIB, therefore PBMCs can also be divided into two categories, such as T cells and FcγR cells. Using antibodies targeting FcγR will also provide the ability to remove non-T cells, whose Fab and Fc regions both bind to FcγR. CD32, also known as FcγRII, is widely expressed in B cells, monocytes, granulocytes, dendritic cells, NK cells, and platelets. Therefore, IgG anti-human CD32 antibodies should be able to remove all FcγRII-expressing cells using the Fab region and other FcγR-carrying cells using the Fc region, along with appropriate levels of conjugated FF, leaving only T cells in the fraction.

[0066] As previously mentioned, in implementation schemes using a single antibody, i.e., a mAb with unique specificity for B cells, to generate naive T cells, if a target for CD4 is added... + or CD8 + The second mAb of T cells, clearly, will produce immature CD4 with the aid of a proper separation system. + or CD8 + Cells. In another embodiment, it is also possible to recover immature CD4 in a single separation process. + or CD8 + Cells and one or the other of those cells labeled with mAb. In other words, a method is provided for obtaining one of those untouched naive subpopulations and another labeled with mAb, said method being carried out in a single magnetic separation or buoyancy separation. For example, if, in one case, a mixture of biotinylated anti-B cell and dethiobiotinylated anti-CD4 mAbs is mixed with PBMCs, then all FcRCs, B cells, and CD4 cells are obtained.+ Cells were magnetically labeled after being added to and incubated with SA solid support. Following magnetic separation, CD8... + Cells will remain in the supernatant or liquid phase, without FcRCs, B cells, and CD4. + Cells. CD8 in suspension + Therefore, they are easily recovered. In this case, the magnetically separated cells will contain "positively selected" CD4. + Cells. These cells can be easily released by adding biotin that disrupts the desulfobiotin:SA bond. If the separation is performed on a device capable of magnetically collecting cells over a large area and on cells in a very homogeneous stratification, as in the system we developed (WO 2016 / 183032 A1), these CD4 cells can be expected to be released. + Good cell recovery rate. If desired, recovery of immature CD4 cells can be achieved through this method by using dethiobiotin-antiCD8 mAb instead of antiCD4 mAb. + Cells. Besides desulfobiotin dissociation via biotin replacement, methods for dissociating the biotin / antibiotin reaction are well known in the art (Lund, G. and Wegmann, T., U.S. Patent No. 5,518,882; Brieden, J. and Dose, C., U.S. Patent No. 20140113315A1). Therefore, there are multiple methods within the above-described schemes for recovering mAb-labeled CD4. + or CD8 + cell.

[0067] In addition to all the embodiments disclosed herein, there is another variable that can be used, which further extends these concepts and methods, making it possible to isolate naive NK cells and even naive B cells. As mentioned above, since all leukocytes except naive T cells express FcγR, and because there is a significant difference in the dissociation constants of high-affinity receptors and low-affinity receptors (Mkaddem S. et al., “Understanding Fc receptor involvement in inflammatory diseases”: From mechanisms to new therapeutic tools. Front Immunol. 10:811, 2019; Chauhan AK, Human CD4 +T-cells: A role for low-affinity Fc receptors. Front Immunol. 7:215, 2016), therefore, optimized negative separation conditions can be achieved by changing the mAb concentration used to label PBMC formulations. We hypothesize that high-affinity FcγRs can be selectively labeled by interacting with Fc on solid supports that aggregate less extensively than those that might require sufficiently strong binding to low-affinity FcγRs. Therefore, it is now possible to isolate FcγRCs carrying only low-affinity FcγRs.

[0068] Another result of forming a complex when a suitable mAb interacts with a multivalent binding surface such as SA-FF and subsequently with FcR is that platelets also carry FcγR. Therefore, when PBMCs are incubated with, for example, sufficiently high concentrations (>2 μg / mL, e.g., 2, 2.5, 3, 3.5, 4, 4.5, 5 μ / mL) of a suitable isotype of biotinylated mAb, and then with SA-FF, platelets also become biotinylated. Therefore, separation using conventional trapping magnetic nanoparticles as described above, or using some other methods employing solid supports or other separation matrices, will also remove platelets from the negative fraction. Since platelet contamination can be a problem even when preparing PBMCs that do not contain most endogenous IgG, they can be removed by their FcγR. Therefore, the present invention not only provides means for producing methods for preparing naive T cells and other cells, but also saves time and money by eliminating the need for any significant processing of blood products before selection begins.

[0069] In addition to all of the above, the principle that FcγRC can be readily eradicated, as disclosed in this article, enables other important clinical strategies. For example, currently, CD34 + Stem cells are isolated using positive selection with anti-CD34 mAbs. The ability to process these cells, starting with untouched or immature cells, for transplantation or gene therapy is needed. With current practices, negative selection can be achieved, but this requires 7-9 mAbs to remove CD34. + All cells except the cellular component are removed. From an economic standpoint, this is not a feasible approach. On the other hand, studies on bone marrow from healthy individuals and fetuses and adults with leukemia have confirmed that FcγR is absent in amorphous progenitor cells CD34. + Cellular expression (Olweus J. et al. CD64 / Fc Gamma RI is a granulo-monocytic lineage marker on CD34) +hematopoietic progenitor cells. Blood.85:2402-13,1995; Aoki Y., et al. Identification of CD34 + and CD34 - Leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood. 125:967-80, 2015. Therefore, uncontaminated CD34 can be achieved using the methods and reagents described herein. + Negative selection of stem cells. As shown below, this result can be achieved using only one mAb, namely biotinylated anti-CD3 mAb.

[0070] The principle of using Fc aggregates on a surface to bind FcγR and platelets can be used for another advantageous purpose. As disclosed, we have found that treatment of PBMCs with RAM-FF can eliminate all monocytes. Furthermore, we have observed that platelets can be eliminated by constructs having aggregated Fc regions on their surface. Therefore, PBMCs and other similar mixtures can be treated with such reagents to produce PBMC formulations and mixtures free of FcR-carrying components. Human IgG binds to surfaces in a manner that brings its Fc fragments close together, which is ideal for binding FcγR-carrying components. The removal of this FcγR carrier can be readily achieved by using magnetic or buoyant particles to pass PBMCs over a densely bound human IgG adsorbent, and simply by labeling such entities, thereby changing their density and enabling centrifugation methods based on the differential density of cell entities.

[0071] In summary, we found that FcγR on FcγRC can be advantageously used for negative selection of naive T cells using only a single antibody that specifically reacts to B cells and the Fc region that binds to FcγR. Two main methods are disclosed, each capable of being performed as a direct labeling method, i.e., using a single key reagent bound to a solid support, or as an indirect labeling method employing a single key reagent and a surface or solid support, such as a common trapping agent, which allows the key reagent to bind to it in a close-packed manner. Various separation methods can be employed.

[0072] Besides direct binding to B cells, we believe the mechanism at play in FcγRC labeling is: (i) when using antibodies against B cells (e.g., anti-CD19 or anti-CD20), FcγRC is labeled with Fc aggregates on a solid support; (ii) in the case of using anti-B cell surface Ig such as mouse anti-human IgG, in fact, mouse anti-human IgG can also bind to immunoglobulins bound to FcγRC and may stabilize those binding reactions through cross-linking with neighboring immunoglobulins, thereby enhancing the binding constant of the FcγR-antibody reaction; (iii) if anti-CD32 is used, the antibody can bind to FcγRII through the Fab complementary site and to all FcγRs through the Fc region. Figure 1 As mentioned above, mechanism "2" is sensitive to free IgG in the system, and therefore is best performed using lower levels of endogenous IgG present in unwashed PBMC samples. Since low-affinity FcγRs interact with closely spaced Fc regions by enhancing their affinity constants, they also appear to play a role in "labeling" FcγRCs. Therefore, by altering the mAb levels bound to PBMCs, only high-affinity FcγRs can be selectively labeled. This ability enables the isolation of naive NK cells and B cells.

[0073] In addition to the above-described method that actually utilizes the “specific binding pair reaction” we observed between FcR and Fc aggregates on the surface, we also disclose an immune-specific targeting method for FcγR as a method for purifying untouched or naive T cells.

[0074] The methods and compositions currently claimed can be used to advantageously prepare cells for CAR T therapy. This invention also provides negative selection of CD34. + The method also has significant therapeutic potential.

[0075] Finally, based on the information provided herein, recombinant molecules can be designed to function as mAbs by generating one or more polypeptide sequences that mimic the FcγR binding region of antibodies that bind to FcγR and molecular entities (combined or independent) that can specifically bind to B cells, or some other specificity according to this application.

[0076] The following materials and methods are provided to assist in carrying out this invention.

[0077] Magnetic Particles - Ferrofluids (FF): All FFs were manufactured by BioMagnetic Solution (State College, PA). BSA / HSA-FF was synthesized by coating FF with bovine serum albumin (BSA, Sigma) or human serum albumin (HSA, Akron Biotech, Boca Raton, FL), resulting in a coated size of approximately 125-130 nm and containing 84% magnetic material. RAM-FF was prepared by conjugating rat anti-mouse IgG1 mAb to BSA or HSA-FF using standard trout reagents and the sulfo-SMCC procedure (Thermo Fisher, Waltham, MA). Similarly, SA-FF was synthesized by conjugating streptavidin (Agilent, Santa Clara, CA) to BSA or HSA-FF. Both RAM-FF and SA-FF are approximately 155-165 nm in size.

[0078] Antibody and flow cytometry analysis: Mouse anti-human IgG polyclonal antibody was obtained from Jackson Laboratory (catalog number 209-005-082). Polyclonal goat anti-human IgG (Fab')2-biotin and Fab-biotin were purchased from Rockland Immunochemicals (Pottstown, PA). mAbs for human CD3, CD19, IgG, CD20, and CD32 were obtained from Absolute Antibody (Boston, MA). Anti-human CD34 and CD56 antibodies were obtained from Biolegend (San Diego, CA). Fluorescent antibodies against CD45-FITC, CD3-PE, CD11b-PEcy5, CD19-PEcy5, CD34-PE, and CD56-PE were obtained from Biolegend. Cells before and after separation were stained with fluorescently conjugated antibodies. CD3, CD11b, and CD19 positive cells were analyzed by gating CD45-positive cells. An isotype control was used to set the analysis gate. easyCyte TM Samples were analyzed using a flow cytometer (Luminex).

[0079] Cell-to-cell separation: Cells used in many experiments are derived from apheresis products that have been centrifuged directly to remove plasma. These cells are washed with PBS containing 0.5% BSA through three centrifugation cycles, with the final precipitate of cells being resuspended in CryoStor-CS10 (Biolife Solutions, Bothell, WA), aliquoted, and frozen. CryoStor-CS10 is a serum-free and protein-free cryopreservation medium for cells. These aliquots are referred to as PBMC-Ig because they are essentially free of endogenous human IgG. Fresh apheresis products have also been used in experiments, washed twice with 1% BSA or HSA in PBS.

[0080] Direct negative cell selection procedure: Following the manufacturer's recommendations, mouse anti-human IgG (MAH-Ig) polyclonal antibodies, primarily of the IgG1 isotype, were conjugated to BSA-FF using Trout reagent and sulfo-SMCC. Conjugation was designed to densely stack the antibody on the FF surface (4000-7000 mAb / particle). Similar conjugation was also performed with anti-human CD19 mAb. Frozen PBMCs were directly diluted with an equal volume of buffer (4% BSA in PBS) and mixed with 15 μg / mL (Fe-based) MAH-Ig-FF and 2-10 x 10⁻⁶ ppm BSA. 7 Incubate at a cell concentration of [number] cells / mL for 20 minutes at room temperature. Since FF readily labels cells via diffusion, no further mixing is required during the 20-minute incubation. At the end of incubation, if necessary, further dilute the cells to 2 x 10⁻⁶ cells / mL. 7 / mL, and separated for 15 minutes in a quadruple magnetic separator. Unseparated cells – the negative fraction – were collected by ablation. The negative fraction (uncontacted cells) was analyzed using flow cytometry with CD45, CD3, and CD11b antibodies using the Guava EasyCyte Plus flow cytometer. CD3 was analyzed. + T cell purity and yield.

[0081] Indirect negative selection procedure: First, incubate cells with biotinylated antibody for 20 minutes, then add SA-FF directly to the cell mixture and incubate for another 15 minutes. Then dilute the cells to 2 x 102 7 / mL and perform magnetic separation.

[0082] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

[0083] Example 1

[0084] Unexposed CD3 was isolated from single-collection products / PBMCs using anti-human IgG antibodies via a direct labeling procedure. +T cells

[0085] The mouse anti-human IgG polyclonal antibody, primarily an IgG1 isotype (mouse anti-human IgG (MAH-Ig)), was obtained from Jackson Laboratory (catalog number 209-005-082) and conjugated to FF using Trout reagent and sulfur-SMCC. The antibody conjugation level on MAH-Ig-FF was approximately 300-500 / μg iron. For these 145nm nanoparticles, this indicates a "dense packing" of antibodies on the particle surface, with each particle containing approximately 4000-7000 mAbs.

[0086] Thawed PBMC-Ig (with endogenous Ig essentially removed) without further treatment was mixed with 15 μg / mL (based on iron concentration) MAH-Ig-FF and 2-10x10 7 Incubate at a cell concentration of [number] cells / mL for 20 minutes at room temperature. Since FF readily labels cells via diffusion, no further mixing is required during the 20-minute incubation. At the end of incubation, optionally dilute the cells to 2 x 10⁻⁶ cells / mL. 7 / mL, and separated for 15 minutes in a quadruple magnetic separator. Unseparated cells – negative fraction – were collected by aspiration. The negative fraction (uncontacted cells) was analyzed by flow cytometry using the Guava EasyCyte Plus flow cytometer, where T cells were identified by staining the cells with anti-CD45-FITC and anti-CD3-PE. See also Figure 2 In multiple experiments, CD3 + The purity of T cells is >95%.

[0087] These results clearly demonstrate that a single antibody with binding specificity to human B cell surface Ig is sufficient to remove most FcγRC and B cells. The 95% purity of T cells is generally higher than that of T cells used for CAR T cell production in major clinical manufacturing tissues.

[0088] Example 2a

[0089] Unexposed CD3 was isolated from single-collection products / PBMCs using biotinylated anti-human IgG antibody and SA-FF. + T cells

[0090] As measured by the HABA assay, polyclonal mouse anti-human IgG (MAH-Ig) was biotinylated (MAH-Ig-biotin) to a level of seven biotin / mAb. SA-FF (145nm) is from BioMagnetic Solutions, State College, PA (catalog number SAFF-109). The concentration was 2-10x10 7PBMC-Ig was incubated with 2-4 μg / mL MAH-Ig-Biotin, primarily composed of IgG1 isoforms, for 20 minutes at room temperature. SA-FF (15 μg / mL) was added to the cell mixture and incubated for another 15 minutes. At the end of incubation, the cells were diluted to 2 x 10⁻⁶ cells / mL. 7 / mL, and then magnetic separation was performed as described above. As in Example 1, cells in the negative fraction were analyzed by fluorescence staining and flow cytometry, and >95% of unexposed or immature CD3 cells were found in multiple experiments. + T cells.

[0091] Given that the common capture agent SA-FF does not react with cells in PBMC-Ig, particularly with any FcγRs in these cells, tFcγRCs were removed in these experiments via one or more of the following mechanisms. First, MAH-Ig-biotin can clearly label B cells when mixed with PBMCs; it can also bind to high-affinity FcγRs, and, if the concentration is sufficient, to low-affinity FcγRs; MAH-Ig-biotin crosslinks occupy the endogenous Ig of FcγRs, and this crosslinking leads to increased binding of those endogenous Igs to their FcγRs due to the multivalent enhancement of the affinity constant. Therefore, MAH-Ig-biotin can biotinylate-label FcγRCs with high affinity. Another reaction that should occur when SA-FF is added to the system after initial antibody incubation is that unbound MAH-Ig-biotin will also simultaneously bind to SA-FF to form a complex capable of binding to FcγRs. Therefore, there are clearly multiple ways in which FcγRCs can be magnetically labeled. In summary: (i) SA-FF binds to biotin on FcγRC through multivalent interactions, resulting in a very strong binding, i.e., affinity; (ii) SA-FF complexes with MAH-Ig-biotin, which strongly interacts with "empty" FcγR or replaces Ig in "filled" FcγR due to the possibility of multivalent attachment; or (iii) MAH-Ig-biotin binds to Ig occupying FcγR and cross-links Ig through multivalent attachment, generating sufficient binding energy to make cells easily labeled by SA-FF.

[0092] Example 2b

[0093] Unexposed CD3 was isolated from apheresis products / PBMCs using a biotinylated anti-human IgG (Fab') 2 fragment. + T cells

[0094] The polyclonal biotinylated (Fab')2 fragment of goat anti-human IgG (FαHIg) was purchased from Rockland Immunochemicals (Pottstown, PA) and used in experiments similar to those described in Example 2a, except that different amounts of human IgG were added to the cells before antibody labeling. The effects of PBMC-Ig pre-incubation with human IgG before adding the biotinylated F(ab')2 fragment of anti-human IgG (FαHIg) are listed in Table 1. With increasing amounts of human IgG, B cell contamination (initially 15% in this formulation) increased significantly, which was expected because the labeled antibody was neutralized by the added human IgG. On the other hand, CD11b... + Cells (initially 29.3%) were significantly less affected. This suggests that complex binding reactions play a role in these experiments. For example, without the addition of human IgG, (Fab')2 is most likely to bind to Ig bound to FcγR, causing these Igs to cross-link and stabilize their interaction with FcγR. Furthermore, as the amount of human IgG added increases, a second phenomenon may occur: (Fab')2 binds to IgG and forms a complex that strongly interacts with FcγR, leading to their biotin labeling and subsequent removal by SA-FF or with SA-FF as part of the Ig-(Fab')2-SA-FF complex. However, increasing the amount of IgG added competes with (Fab')2 for interaction with B cell surface IgG and affects B cell eradication.

[0095] Table 1. Effects of adding human IgG before FαHIg on the purity and yield of isolated naïve T cells.

[0096] [hIgG] (μg / mL) during pre-incubation none 125 250 Before separation <![CDATA[CD3 + Cell purity, % 95.3 88.7 84.3 54.94 <![CDATA[CD11b + Cells, % 0.7 1.2 2.4 29.28 <![CDATA[B cells (CD19 + ), %]]> 0.9 8.3 13.4 15.15 <![CDATA[CD3 + Cell yield, % 57.0 73.0 78.9

[0097] When nearly identical experiments were performed using biotinylated anti-CD19 mAb, highly purified naive T cells were produced. Pretreatment with human IgG had little or no effect in these experiments, confirming our hypothesis regarding the interaction mechanism.

[0098] Example 3

[0099] Unexposed CD3 was isolated from apheresis products using biotinylated anti-human CD32 and SA-FF. + T cells

[0100] Because FcγR can be used to remove FcγRC, as shown in the examples above, it should be possible to remove all such cells with antibodies bound to FcγR by combining antibody-epitope reactions and magnetic labeling of aggregated Fc-mediated FcRC in the following manner. Anti-CD32 antibodies bind to FcγRII, which is widely expressed on leukocytes (e.g., B cells / monocytes / granulocytes / platelets) but not on T cells. This antibody can be used to label all cells carrying FcγRII via antibody-epitope reactions, including any B cells in the formulation. Unbound biotinylated anti-CD32 antibodies also bind to the SA-FF reaction, causing Fc to aggregate on FF, which also affinity-binds FcγRC. This method can label all γ-class FcRs, using anti-CD32 antibodies in one case and anti-CD32 antibodies bound to the capture surface introduced into the system in another. After incubation in a suitable separation device, a high-purity suspension of naive T cells is obtained. To demonstrate the efficiency of this method, IgG-based biotinylated mouse mAbs with affinity for human CD32 were used. The concentration was 2-10 x 10⁻⁶. 7 PBMCs were incubated with 2 μg / mL of biotinylated mouse anti-human CD32 mAb of IgG1 isotype at room temperature for 15 minutes—providing sufficient unbound mAb for subsequent reactions. SA-FF was added to the cell mixture and incubated for another 15 minutes. Effective labeling of FcRγC was achieved either by antibody-epitope reaction or by aggregation of Fc on SA-FF. After cell isolation, uncontaminated or immature CD32 was found in the negative fraction. + The purity of T cells is >92%.

[0101] Example 4

[0102] CD4 negative selection was performed using two antibodies from apheresis products or PBMCs. + or CD8 + T cells

[0103] Based on the preceding embodiments, a method for preparing naive CD4 was designed. + or CD8 + The cellular approach includes an additional mAb targeting either CD4 or CD8, depending on the type of T cells to be negatively selected. Using CD4 + The negative selection of naive cells illustrates that both mAbs will be immobilized on FF, with one or both reacting with FcγR, i.e., the correct subclass / isotype. Therefore, if Example 1 is modified by conjugating anti-human IgG and anti-CD8 mAb to FF, such conjugates should eliminate all FcγRC, B cells, and CD8 in the absence of significant competitive IgG. + Cells, leaving untouched CD4 in the negative fraction. +Cells. To obtain immature CD8 + For cells, the second mAb that is fixed will be anti-CD4, not anti-CD8. Therefore, the negative fraction will contain CD8. + cell.

[0104] In another approach, when using a common capture agent such as SA-FF, the same results can be achieved using two biotinylated mAbs. Therefore, CD8 can be labeled by incubating PBMCs with biotinylated anti-CD8 and anti-B cell-specific mAbs. + Cells, B cells, and FcRC can be used to produce naïve CD4. + Cells. Magnetic separation using SA-FF produces negatively selected CD4 cells. + Cells. Based on the purity species reported in this paper, CD4 is expected to be present. + Cell purity will reach a medium-to-high level of 90%. This was experimentally confirmed using anti-CD19-biotin and anti-CD8-biotin combined with SA-FF, yielding CD4 cells with a purity >92%. + cell.

[0105] Example 5

[0106] CD4 is recovered using two mAbs in a single magnetic separation. + and CD8 + cell

[0107] Based on CD4 at a defined ratio + and CD8 + Given the clinical importance of cell-derived CAR T constructs, efficient methods for achieving such isolation are necessary. Using the procedures described herein, it is reasonable to expect that the following two mAb-specific protocols can accomplish this task. One strategy for achieving this isolation is the complete removal of CD4 from PBMC-Ig. + All extracellular cells will release CD4 + Cells remained in the negative fraction, and CD8 was then recovered from the positively selected fraction via a simple extraction process. + cell.

[0108] For example, PBMCs, which essentially lack IgG, can interact with biotinylated mAbs that interact with FcγR, such as B cell-specific isotype IgG. 1、 IgG2 or IgG3, as well as desulfurized biotinylated mAbs of IgG subclasses and CD8 + Cell-specific non-FcR-binding antibodies were incubated together. Therefore, all cells carrying FcγR were labeled with biotin-tagged anti-B cell mAbs, while CD8+ was also inoculated. +Cells will be labeled with desulfurized biotinylated mAbs. After adding a common trapping agent, such as SA-FF, binding and dissociation of CD8... + Cells and all FcγRC will be positively separated, while the remaining negatively selected naive CD4 cells will be separated. + T cells are readily retrievable. Based on the highly significant difference in the binding constants of biotin and dethiobiotin to SA (Hirsch JD, et al., Analytical Biochemistry. 308:343–357, 2002), it should be possible to release CD8 from SA-FF via incubation with biotin. + In cells, biotin will replace desulfobiotin mAbs at their SA binding sites. Besides the desulfobiotin dissociation via biotin substitution, methods for dissociating the biotin-antibiotin reaction are well known in the art (Lund, G. and Wegmann, T., U.S. Patent No. 5,518,882; Brieden, J. and Dose, C., U.S. Patent No. 20140113315A1). Therefore, there are multiple methods within the above-described schemes for recovering mAb-labeled CD4. + or CD8 + cell.

[0109] To recover CD8 from SA-FF or other suitable dissociable binding pairs + For cells, it is advantageous to use a magnetic separation device, which separates magnetically labeled cells by scattering them on a collection surface with sufficient area and magnetic gradient characteristics so that they do not clump together. If this is achieved, as with the magnetic separation system disclosed in patent publication WO2016 / 183032A1, CD8 + Cells can be gently extracted while they are magnetically immobilized on the collection surface. Alternatively, magnetically captured cells can be suspended in biotin (in the case of the SA-Biotin system), resulting in release. The mixture is then separated again, leaving easily recoverable suspensions of CD8. + cell.

[0110] Example 6

[0111] Uncontaminated B cells were isolated from apheresis products by indirect labeling with two mAbs.

[0112] Based on the results of the foregoing embodiments, we successfully produced naive B cells using these methods. In this method, we utilized the fact that FcγR expressed on leukocytes has different binding affinities. For example, monocytes, macrophages, dendritic cells (DCs), and granulocytes express high-affinity FcγR (FcγRI), while B cells and NK cells express low-affinity FcγR (FcγRIIB for B cells, and FcγRIIC and FcγRIIIIA for NK cells). Although high-affinity FcγR is insensitive to valence, low-affinity FcγR preferentially binds to multimeric antibodies. Since B cells express low-affinity FcγR (FcγRIIB), reducing the mAb concentration used in the labeling step and adjusting the SA coating on the FF should only promote high-affinity reactions, thereby preferentially labeling cells carrying high-affinity FcγR in the reaction mixture, such as monocytes, granulocytes, and DCs, by Fc binding to the mAb used for targeting. Due to CD3... + Cellular and NK cells also need to be targeted for removal, therefore two mAbs suitable for this purpose can be used, including but not limited to anti-CD3 and anti-CD16. PBMC-Ig can be incubated with biotinylated anti-CD3 (approximately 0.15 μg / mL) and anti-CD16 (approximately 0.05 μg / mL) for 15 to 20 minutes to achieve appropriate labeling levels. All unwanted cell types can then be removed using common trapping magnetic nanoparticles or some other suitable reagents well known in the art. These methods enable the recovery of highly pure, untouched B cells.

[0113] Example 7

[0114] Unexposed NK cells were isolated from apheresis products or PBMCs using two antibodies.

[0115] 5-10% of PBMCs are NK cells, which are a key component of revolutionary CAR-T immunotherapy, providing one of the most potent anticancer agents known to date (Shimasaki N., et al., Nature Reviews Drug Discovery, 2020, 19:200–218). Isolation of naive NK cells for genetic engineering using cancer antigens constitutes a crucial initial step in CAR-T. Based on the same concept detailed in Example 6, isolated uncontaminated NK cells can be obtained in the absence of substantially competing IgG by limiting the mAb concentration and SA density on FF particles during the labeling reaction. In one approach, a combination of biotinylated mouse anti-human B cell antibody (e.g., anti-CD19 mAb) and biotinylated mouse anti-human CD3 antibody is used, with at least one reagent being an IgG1 isotype. The antibodies are added at a total concentration of approximately 0.2 μg / mL (0.15 μg / mL anti-CD3 mAb and 0.05 μg / mL anti-B(CD19) mAb). Under these antibody-limited conditions, only high-affinity FcγR binding occurs. After incubation with mAb at approximately the above concentration, SA-FF is added, and separation is performed as described in Example 2. As the mAb concentration decreases, T cells, B cells, and those with high affinity for FcγRC will be magnetically separated, thereby leaving NK cells in the negative fraction.

[0116] Example 8

[0117] Unexposed CD34 was isolated by negative selection. + hematopoietic stem cells

[0118] In the widely used current scheme, CD34 + Stem cells are isolated using positive selection with anti-CD34 mAbs. The ability to process these cells, starting with untouched or immature cells, is needed for transplantation or gene therapy. With current practices, negative selection can be achieved, but this requires 7-9 mAbs to remove CD34. + All cells except the cellular component are removed. From an economic standpoint, this is not a feasible approach. On the other hand, studies on bone marrow from healthy individuals and fetuses and adults with leukemia have confirmed that FcγR is absent in amorphous progenitor cells CD34. + Cellular expression (Olweus J. et al. CD64 / Fc Gamma RI is a granulo-monocytic lineage marker on CD34) +hematopoietic progenitor cells. Blood.85:2402-13,1995; Aoki Y., et al. Identification of CD34 + and CD34 - leukemia-initiating cells in MLL-rearranged human acute lymphoblastic leukemia. Blood. 125:967-80, 2015).

[0119] Therefore, it is necessary to start by selecting an appropriate mAb to completely remove CD34 from PBMCs. + For all cells except immature cells, the following strategy can be applied: Incubate biotinylated mAb-anti-CD3 with PBMCs for approximately 20 minutes to label all T cells and FcγR-expressing cells. Negative selection of unlabeled cells can be accomplished using SA on a solid support such as FF. The supernatant from this magnetic separation will be enriched with CD34. + Cell populations are separated from solution magnetically because T cells, B cells, and other FcγRCs (including platelets). In some embodiments, the donor PBMCs have been treated with G-CSF to induce hematopoietic stem cell migration from the bone marrow into the peripheral blood.

[0120] In this implementation, it is important to thoroughly label all cells to be removed by providing the system with sufficient biotinylated mAb and adequate incubation time, as approximately 98-99% of nucleated cells need to be removed. The cell concentration is approximately 1 x 10⁻⁶ cells / mL. 8 At a concentration of 2 μg / mL, incubating with a high-affinity mAb in a cell suspension for approximately 20 minutes should be sufficient. In this case, there will be approximately 80,000 mAbs / cell, which should be more than enough for labeling and subsequent removal of such cells.

[0121] With such a large proportion of cells removed, there is a possibility that the required CD34 may be entrained. + This poses a risk to cells. Therefore, it is recommended to keep the total cell concentration below approximately 3 x 10⁻⁶. 7 Total cells / mL and possibly as low as 5 x 10⁻⁶ 6This type of separation is performed when the total number of cells is [number missing]. Based on our experience, we found that adding 1% sucrose significantly reduces entrainment. Furthermore, when using magnetic separation, it is recommended to remove unwanted cells by large-area separation to minimize retention. To achieve this, a separation system such as the one disclosed by Liberti et al. in WO2016 / 183032A1 is recommended. This system not only minimizes the collection of clumps of cells but also provides a method for removing entrained cells through a process called "meniscus scrubbing" (a gentle method for removing entrained cells).

[0122] While certain preferred embodiments of the invention have been described and specifically illustrated above, this does not mean that the invention is limited to these embodiments. All patents, patent applications, and publications cited herein are expressly incorporated herein by reference in their entirety for all purposes. Various modifications may be made to the invention as set forth in the claims.

Claims

1. A method for isolating immature target CD3 from a peripheral blood mononuclear cell (PBMC) formulation containing at least T cells and B cells. + A direct method for T-cell fractionation, wherein the formulation is substantially lacking in endogenous or added IgG and targets CD3. + The method comprises: T cells essentially lack Fc receptors on their surface. (a) Introducing into the PBMC formulation a single immunoactive trapping agent comprising an anti-B cell antibody or a functional fragment thereof, the trapping agent having a Fab region for binding to epitopes on B cells and an Fc region for binding to Fcγ receptors on non-target cells, the trapping agent being operatively linked to a ferrofluid comprising magnetically responsive particles, the trapping agent binding to both non-target cells carrying Fcγ receptors and B cell epitopes present in the formulation, thereby forming, upon binding, a magnetic cluster of cells carrying Fcγ receptors selected from B cells, monocytes, granulocytes, macrophages, dendritic cells, NK cells and platelets; (b) In a magnetic separator, magnetic clusters of cells and B cells carrying Fcγ receptors from the formulation are combined with the naïve target CD3. + T cell fractionation; and (c) Recovering the naive target CD3 + T cell classification.

2. A method for isolating immature target CD3 from a peripheral blood mononuclear cell (PBMC) formulation containing at least T cells and B cells. + An indirect method for T-cell fractionation, wherein the formulation is substantially lacking in endogenous or added IgG and targets CD3. + The method comprises: T cells essentially lack Fc receptors on their surface. (a) Introducing a capture agent comprising a first binding pair member of a specific binding pair into the PBMC formulation, the capture agent having a Fab region and an Fc region, which bind to both epitopes present in the formulation on non-target cells and B cells carrying Fcγ receptors. (b) Under the condition that a specific binding pair is formed between the first binding pair member and the second binding pair member, the formulation of step (a) is contacted with a ferrofluid containing magnetically responsive particles operatively linked to the second binding pair member to form a magnetic cluster of cells carrying Fcγ receptors selected from B cells, monocytes, granulocytes, macrophages, dendritic cells, NK cells and platelets. (c) In a magnetic separator, magnetic clusters containing bound cells are combined with the naïve target CD3. + T cell fractionation; and (d) Recover naive target CD3 + T cell classification.

3. The method according to claim 1 or 2, wherein the Fab region binds a B-cell epitope selected from CD19, CD20, IgG and CD32.

4. The method according to claim 1 or 2, wherein the antibody in the trapping agent is a mouse or human monoclonal IgG antibody and comprises an Fc region that is bound by human FcγR.

5. The method according to any one of claims 2 to 4, wherein the first binding pair member and the second binding pair member are selected from biotin-streptavidin, receptor-ligand, agonist-antagonist, lectin-carbohydrate, avidin-biotin, biotin analog-avidin, desulfobiotin-streptavidin, desulfobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.

6. The method of claim 1 or 2, wherein the trapping agent comprises a ferrofluid having magnetically responsive particles operably linked to a rat anti-mouse IgG antibody or a mouse anti-human IgG antibody.

7. The method according to claim 1 or 2, wherein each antibody comprises 3-7 biotin molecules.

8. The method of claim 2, wherein the trapping agent is a first immunologically active trapping agent, and step (a) further comprises reacting with CD8. + A second immune-active trapping agent bound to T cells, each of the first and second immune-active trapping agents being operatively linked to magnetically responsive particles present in a ferrofluid; wherein, following contact in step b) and separation in step c), the separated magnetic clusters contain cells bound to the trapping agent, selected from B cells, monocytes, granulocytes, macrophages, dendritic cells, NK cells, platelets, and CD8+ cells. + T cells; and (d) Recycle CD3 subclasses that are in a basically immature state. + T cell classification, which is also CD4 + T cells.

9. The method of claim 2, wherein the trapping agent is a first immunologically active trapping agent, and step (a) further comprises reacting with CD4. + A second immune-active trapping agent bound to T cells, each of the first and second immune-active trapping agents being operatively linked to magnetically responsive particles present in a ferrofluid; wherein, following contact in step b) and separation in step c), the separated magnetic clusters contain cells bound to the trapping agent, selected from B cells, monocytes, granulocytes, macrophages, dendritic cells, NK cells, platelets, and CD4+ cells. + T cells; and (d) Recycle CD3 subclasses that are in a basically immature state. + T cell classification, which is also CD8 + T cells.

10. The method according to claim 1 or 2, wherein the ferrofluid comprises 4,000-7,000 monoclonal antibodies / particles.

Images