RU2777989C2 - Oligomeric reagents in form of particles and their application methods - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: oligomeric reagent is presented, including oligomeric reagents of streptavidin or streptavidin mutein, where oligomeric reagents in the form of particles contain a set of agent binding sites, which are multimerized by reversible binding to the oligomeric reagent in the form of particles, thus creating a multimerized oligomeric reagent in the form of particles, having stimulating agents multimerized on it. A method for stimulation of cells is also presented, wherein the method includes incubation of a cell composition containing target cells in the presence of the multimerized oligomeric reagent in the form of particles.

EFFECT: invention allows for the production of reagents of a cell culture with improved characteristics for the stimulation of a cell population and further use in adoptive cell immunotherapy or anticancer therapy.

80 cl, 15 dwg, 5 tbl, 6 ex

Description

Link to related applications

This application claims priority under U.S. provisional application 62/491245 entitled "OLIGOMERIC PARTICLE REAGENTS AND METHODS OF USE THEREOF" filed April 27, 2017, the contents of which are incorporated herein by reference in their entirety.

Including a sequence listing via a link

This application is filed along with the Sequence Listing in electronic format. The sequence list is provided as a file named 735042008540SeqList.TXT, created on April 26, 2018, which is 110426 bits in size. The sequence listing information in electronic format is incorporated herein by reference in its entirety.

The technical field to which the invention belongs

The present disclosure provides oligomeric reagents, including streptavidin or streptavidin mutein oligomeric reagents, and their compositions and methods for preparing oligomeric reagents, including methods for reliably obtaining oligomeric reagents in the form of particles of the desired size. In some cases, the reagents are particulate oligomeric reagents containing multiple agent binding sites, and thus one or more agents, for example, one or more selective agents or stimulating reagents, multimerize by reversibly binding to the particulate oligomeric reagent, for example, thus creating a multimerized particulate oligomeric reagent having stimulating or selective agents multimerized thereon. The present disclosure also provides methods of using oligomeric reagents for incubation or culture, for example, to stimulate reproduction (proliferation), activation, costimulation and/or survival, cell composition, such as a population of lymphocytes. In some aspects, the disclosure provides methods and reagents for stimulating, for example, reproduction (proliferation), survival or persistence, activation, costimulation or other action (for example, affinity selection) of cell populations, which include binding agents to a molecule on the surface of cells, thus providing cells with one or more signals.

State of the art

There are various strategies to stimulate populations of T cells in vitro, including the expansion of antigen-specific T cells in vitro for use in adoptive cell immunotherapy or cancer therapy, in which infusions of such T cells have been shown to have antitumor activity in a tumor-bearing recipient or for use in the treatment of viral infections. Improved strategies are needed to expand populations of cells in vitro, including for research, diagnostic and therapeutic purposes.

Essence of the Invention

Provided herein are particulate oligomeric reagents containing a plurality of streptavidin or streptavidin mutein molecules, wherein the size of the particulate oligomeric reagent is i) a radius, e.g., a hydrodynamic radius greater than 25 nm, ii) a molecular weight of at least 5× 10 ⁶ g/mol; and/or (iii) at least 100 tetramers of streptavidin or streptavidin mutein per oligomeric particulate reagent.

In specific embodiments of any of the particulate oligomeric reagents provided herein, the streptavidin or streptavidin mutein molecules bind or are capable of binding to biotin, a biotin analog (e.g., destiobiotin, iminobiotin), or a streptavidin-binding peptide (e.g., Strep-tagII (WSHPQFEK, SEQ ID NO:8)). In some embodiments of any of the oligomeric particulate reagents provided herein, the streptavidin or streptavidin mutein molecules reversibly bind or are capable of reversibly binding to biotin, a biotin analog, or a streptavidin binding peptide (e.g., Strep-tagII (WSHPQFEK, SEQ ID NO:8)). In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules, wherein the streptavidin mutein molecules comprise the amino acid sequence Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or lle ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44-47 with reference to positions in streptavidin in the amino acid sequence shown in SEQ ID NO: 1.

In particular embodiments of any of the particulate oligomeric reagents provided herein, the streptavidin or streptavidin mutein molecules bind or are capable of binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and/or a biologically active fragment thereof. In some embodiments of any of the particulate oligomeric reagents provided herein, the streptavidin or streptavidin mutein molecules reversibly bind or are capable of reversibly binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and/or a biologically active fragment thereof. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules, wherein the streptavidin mutein molecules comprise the amino acid sequence Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or lle ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44-47 with reference to positions in streptavidin in the amino acid sequence shown in SEQ ID NO: 1.

In specific embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that comprise: a) an amino acid sequence as set forth in any of SEQ ID NOs: 3-6, 27, 28, 60 or 61; b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to any SEQ ID NO: 3-6, 27, 28, 60 or 61 and contain the amino acid sequence corresponding to Va1⁴⁴-Thr⁴⁵-ala⁴⁶-Arg⁴⁷or lle⁴⁴-Gly⁴⁵-ala⁴⁶-Arg⁴⁷, and/or bind reversibly to biotin, a biotin analog, or a streptavidin-binding peptide; and/or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any SEQ ID NO: 3-6, 27, 28, 60 or 61 and contain the amino acid sequence corresponding to Va1⁴⁴-Thr⁴⁵-ala⁴⁶-Arg⁴⁷or lle⁴⁴-Gly⁴⁵-ala⁴⁶-Arg⁴⁷, and/or binds to biotin or its biologically active form, biotin analogue or mutein or their biologically active fragment or streptavidin-binding peptide, functional fragment a) or b) that binds to biotin or its biologically active form, biotin analogue or mutein or their biologically active fragment or streptavidin-binding peptide and/or reversibly associated with biotin, biotin analogue or streptavidin-binding peptide; or c) a functional fragment of a) or b) that binds to biotin, a biotin analog, or a streptavidin-binding peptide. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that comprise the amino acid sequence set forth in SEQ ID NO: 6 or 61. In some embodiments of any of the oligomeric particulate reagents. of the particulate reagents provided herein, the streptavidin mutein molecule further comprises an amino acid substitution or substitutions at position corresponding to 117, 120 and/or 121 with reference to positions in streptavidin in the amino acid sequence shown in SEQ ID NO: 1.

In specific embodiments of any of the particulate oligomeric reagents provided herein: the amino acid substitution or substitutions are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, or Phe121; or the amino acid substitution or substitutions are selected from one or more of Glu117, Gly120 or Tyr121; or amino acid substitutions are selected from Glu117, Gly120 or Tyr121. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that contain: a) the amino acid sequence shown in SEQ ID NO: 27 or 28; b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to SEQ ID NO: 28 and contains the amino acid sequence corresponding to Va1 ⁴⁴ , Thr ⁴⁵ , Ala ⁴⁶ , Arg ⁴⁷ , Glu ¹¹⁷ , Gly ¹²⁰ and Tyr ¹²¹ and/or binds to biotin or a biologically active fragment, analogue or mutein of biotin or their biologically active fragment or streptavidin-binding peptide; or c) a functional fragment of a) or b) that binds to biotin or a biologically active fragment, analog or mutein of biotin or their biologically active fragment or streptavidin-binding peptide.

In particular embodiments of any of the particulate oligomeric reagents provided herein: the amino acid substitution or substitutions are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121, or Phe121; or the amino acid substitution or substitutions are selected from one or more of Glu117, Gly120 or Tyr121; or amino acid substitutions are selected from Glu117, Gly120 or Tyr121. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that contain: a) the amino acid sequence shown in SEQ ID NO: 27 or 28; b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to SEQ ID NO: 28 and contains the amino acid sequence corresponding to Va1 ⁴⁴ , Thr ⁴⁵ , Ala ⁴⁶ , Arg ⁴⁷ , Glu ¹¹⁷ , Gly ¹²⁰ and Tyr ¹²¹ and/or binds reversibly to biotin , a biotin analog, or a streptavidin-binding peptide; or c) a functional fragment of a) or b) that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide.

In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent is bound or capable of being associated with one or more agents via a binding partner, wherein the one or more agents comprise a binding partner such as biotin, a biotin analog; or a streptavidin-binding peptide. In particular embodiments of any of the particulate oligomeric reagents provided herein, one or more agents comprise a binding partner, wherein the binding partner is capable of binding to one or more binding sites on the particulate oligomeric reagent. In some embodiments of any of the particulate oligomeric reagents provided herein, the binding partner comprises a streptavidin binding peptide or biotin or a biotin analog. In some embodiments of any of the particulate oligomeric reagents provided herein, the binding partner comprises a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His- Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19) In specific embodiments, any of the oligomeric particulate reagents provided herein, one or more agents bind or are able to further bind to a molecule expressed on the surface of a target cell.In some embodiments of any of the oligomeric particulate reagents provided herein, one or more agents contain antibody, optional specifically a Fab or Nanobody®, such as a single domain antibody (sdAb).

In some embodiments of any of the particulate oligomeric reagents provided herein, the one or more agents is a receptor binding agent that binds or is capable of binding to a receptor expressed on the surface of a target cell. In specific embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, thereby inducing or modulating a signal in the target cell. In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3. In some embodiments of any of the particulate oligomeric reagents provided herein, the stimulatory agent is a first receptor binding agent and the particulate oligomeric reagent comprises a second receptor binding agent, wherein the second receptor binding agent is capable of specifically binding with a second molecule on the surface of the target cell, and this binding to the second molecule is optionally capable of inducing or modulating a signal in the target cells.

In particular embodiments of any of the particulate oligomeric reagents provided herein, the second receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family, or is a TNF family receptor, cytokine receptor, chemokine the receptor is or is or contains an adhesion molecule or a factor that causes cytokine production, chemokine production and/or expression of an adhesion molecule. In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family, or is a TNF family receptor, cytokine receptor, chemokine receptor or is or contains an adhesion molecule or a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1) , CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In particular embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor and the cytokine receptor is selected from IL-2R, IL-1R, IL-15R , IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7 , CCR9, CXCR1, CXCR3 and CXCR4.

In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand that specifically binds to the receptor cytokine or chemokine.

In particular embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein the ligand specifically binds IL-2R, IL- 1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or he represents their biologically active fragment.

In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein the ligand specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof. In some embodiments of any of the particulate oligomeric reagents provided herein, the receptor binding agent (second receptor binding agent) is an adhesion molecule and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1) , CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is their biologically active fragment.

In particular embodiments of any of the particulate oligomeric reagents provided herein, one or more of the agents is a selective agent, wherein the selective agent binds or is able to bind to a selectable marker that is expressed on the surface of the target cell. In some embodiments of any of the particulate oligomeric reagents provided herein, the target cell is an immune cell. In some embodiments of any of the particulate oligomeric reagents provided herein, the target cell is a lymphocyte or an antigen presenting cell. In specific embodiments of any of the particulate oligomeric reagents provided herein, the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell. In some embodiments of any of the particulate oligomeric reagents provided herein, the target cell is a T cell. In some embodiments of any of the particulate oligomeric reagents provided herein, the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

In specific embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent has a radius greater than 25 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, or greater. than 90 nm. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent has a radius between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm or between 90 nm and 110 nm, inclusive, or 90 nm ± 15 nm or 95 nm ± 20-25 nm. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent has a radius of less than 150 nm. In particular embodiments, the radius is the hydrodynamic radius.

In particular embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent has a molecular weight of at least 1×10 ⁷ g/mol, at least 5×10 ⁷ g/mol, or at least 1×10 ⁸ g/mol. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent has a molecular weight between 1×10 ⁶ g/mol and 1×10 ¹⁰ g/mol, between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol, between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g/mol mol and 2×10 ⁸ g/mol. In some embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises at least 100 streptavidin tetramers or streptavidin mutein, at least 500 streptavidin tetramers or streptavidin mutein, at least 1000 streptavidin tetramers, or a streptavidin mutein, at least 1500 streptavidin tetramers or streptavidin mutein, or at least 2000 streptavidin tetramers or streptavidin mutein.

In particular embodiments of any of the particulate oligomeric reagents provided herein, the particulate oligomeric reagent comprises between 100 and 50,000 tetramers of streptavidin or streptavidin mutein, between 1,000 and 20,000 tetramers of streptavidin or streptavidin mutein, between 1,000 and 10,000 tetramers of streptavidin or streptavidin mutein or between 2000 and 5000 tetramers of streptavidin or streptavidin mutein. In some embodiments of any of the particulate oligomeric reagents provided herein, a plurality of streptavidin molecules or streptavidin muteins contain lysine residues, with less than 20%, 10%, 5%, 1% of the lysine residues containing an N-substituted iminothiolane.

This document provides a composition containing one or more oligomeric reagents in the form of particles. In some embodiments of any of the compositions provided herein, the one or more particulate oligomeric reactants is a plurality of particulate oligomeric reactants. In particular embodiments of any of the compositions provided herein, the plurality of particulate oligomeric reactants have i) an average radius greater than 70 nm; ii) an average molecular weight of at least 1×10 ⁸ g/mol; and/or iii) an average amount of streptavidin or streptavidin tetramers per particulate oligomeric reagent of at least 2000 and/or iv) a radius size distribution in which at least 95% of the plurality of particulate oligomeric reagents have a radius between 10 nm and 150 nm. In some embodiments, the radius size distribution is a hydrodynamic radius size distribution.

In some embodiments of any of the compositions provided herein, the plurality of particulate oligomeric reactants have an average radius greater than 25 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm or more than 100 nm. In some embodiments of any of the compositions provided herein, the plurality of particulate oligomeric reagents have an average radius between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 110 nm, or between 90 nm and 110 nm, inclusive, or 90 nm ±15 nm or 95 nm ± 20-25 nm. In particular embodiments of any of the compositions provided herein, at least 95% of the plurality of particulate oligomeric reactants have a radius between 50 and 150 nm, between 70 nm and 140 nm, between 80 nm and 120 nm, between 80 nm and 115 nm, between 80 nm and 100 nm, between 90 nm and 110 nm and/or between 100 nm and 120 nm.

In some embodiments of any of the compositions provided herein, at least 95% of the particulate oligomeric reactants have a radius within ±50%, ±25%, ±20%, ±15%, ±10%, and/or ± 5% of the mean and/or median radius of a plurality of particulate oligomeric reactants. In some embodiments of any of the compositions provided herein, a plurality of particulate oligomeric reactants have an average radius between 80 nm and 115 nm, and wherein at least 95% of the particulate oligomeric reactants have a radius within ± 25% medium radius. In particular embodiments of any of the compositions provided herein, the plurality of particles have an average molecular weight between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g/mol and 2×10 ⁸ g /mol, inclusive.

In some embodiments of any of the compositions provided herein, the plurality of particulate oligomeric reagents comprises an average amount of streptavidin or streptavidin tetramers per particulate oligomeric reagent of at least 100, at least 500, at least 1000, at least 1500 or at least 2000. In some embodiments of any of the compositions provided herein, the plurality of particulate oligomeric reagents comprises an average amount of streptavidin or streptavidin tetramers per oligomeric particulate reagent between 100 and 50,000, between 1000 and 20,000, between 1000 and 10000 or between 2000 and 5000, always inclusive.

In specific embodiments of any of the compositions provided herein, the average radius of the plurality of oligomeric particles does not increase by more than 25% when stored at or below -80°C, at or below -20°C, and/or at or about or below 4°C for at least 1 week. In some embodiments of any of the compositions provided herein, the average radius of the plurality of oligomeric particles does not increase by more than 10% when stored at or below 4° C. for at least one week. In some embodiments of any of the compositions provided herein, the average radius of the plurality of oligomeric particles does not increase by more than 10% when stored at or below 4° C. for at least 3 weeks. In particular embodiments of any of the compositions provided herein, the average radius of a plurality of oligomeric particles does not increase by more than 10% when stored at or below 4° C. for at least 9 weeks, at least 27 weeks, or at least least 46 weeks. In specific embodiments, the average radius of a plurality of oligomeric particles does not increase by more than 10% when stored at or below -20°C, -30°C, -40°C, -50°C, -60°C, -70° C or -80°C for at least 1 week, 3 weeks, 9 weeks, 27 weeks or 46 weeks.

This document provides methods for preparing a particulate oligomeric reagent containing streptavidin or a streptavidin mutein, the method comprising: incubating a plurality of activated streptavidin or streptavidin mutein molecules containing a thiol-reactive functional group capable of reacting with a thiol functional group, and a plurality of thiolated streptavidin molecules or a streptavidin mutein containing one or more thiol functional groups, thereby creating oligomeric particles of streptavidin or streptavidin mutein; separating oligomeric particles from monomeric and/or smaller oligomeric molecules; and contacting the oligomeric particles with a stabilizing agent, thereby obtaining a particulate oligomeric reagent.

In some embodiments of any of the methods provided herein, a plurality of activated streptavidin or streptavidin mutein molecules are created by incubating a first plurality of streptavidin or streptavidin mutein molecules with an activating agent that is capable of converting one or more amines to a thiol-reactive functional group. In specific embodiments of any of the methods provided herein, a plurality of thiolated streptavidin or streptavidin mutein molecules is created by incubating a second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent that adds or is capable of adding a thiol functional group to one or more lysine residues.

Also provided herein is a method for preparing particulate oligomeric reagents, the method comprising: (a) incubating a first plurality of streptavidin or streptavidin mutein molecules with an activating agent under conditions to convert one or more amines to a thiol-reactive group capable of reacting with a thiol functional by a group, thereby generating a plurality of activated streptavidin or streptavidin mutein molecules; (b) incubating a second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent that adds or is capable of adding a thiol functional group to one or more lysine residues, thereby creating a plurality of thiolated streptavidin or streptavidin mutein molecules; and (c) incubating a plurality of activated streptavidin or streptavidin mutein molecules with a plurality of thiolated streptavidin or streptavidin mutein molecules, thereby obtaining a particulate composition comprising the particulate oligomeric reagents; wherein the method is carried out under conditions in which, at the start of incubation in (c), the plurality of thiolated streptavidin or streptavidin mutein molecules are such that, on average, at least 60% of the lysines contain a thiol functional group, and/or, on average, at least 10 lysines on a thiolated streptavidin tetramer or streptavidin mutein contain a thiol functional group.

Some embodiments further include separating the particulate oligomeric reactants from monomeric and/or smaller oligomeric streptavidin or streptavidin mutein molecules. In some embodiments of any of the methods provided herein, the incubation of the first plurality of streptavidin or streptavidin mutein molecules with an activating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to activating reagent between 1:1 and 1:10. In some embodiments of any of the methods provided herein, the incubation of the first plurality of streptavidin or streptavidin mutein molecules with an activating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to activating reagent of 1:2 ± 2%. In specific embodiments of any of the methods provided herein, the activating agent comprises a heterobifunctional crosslinker. In some embodiments of any of the methods provided herein, the activating agent comprises sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo SMCC) and/or succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH) . In some embodiments of any of the methods provided herein, the thiol-reactive functional group is a haloacetyl group, maleimide group, aziridine group, acryloyl group, arylizing agent, vinyl sulfone group, pyridyl disulfide, TNB-thiol, or a disulfide reducing agent. In specific embodiments of any of the methods provided herein, the thiol-reactive functional group is a maleimide group. In some embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at neutral pH.

In some embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a pH between 6.8 and 7.5. In specific embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a pH between 7.0 and 7.4, optionally at or about 7.2. In some embodiments of any of the methods provided herein, the first set of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a temperature between 4°C and 39°C. In some embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at room temperature, optionally between 20°C and 25°C, optionally at about 23°C or about 24°C.

In specific embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated for 15 minutes to 6 hours or 30 minutes to 2 hours, always inclusive. In some embodiments of any of the methods provided herein, the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated for 45 minutes to 1.5 hours, inclusive, optionally within or about 1 hour.

In some embodiments of any of the methods provided herein, the incubation of a second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent is carried out at a molar ratio of thiolating reagent to each primary amine between 10:1 and 1:1, inclusive, per streptavidin or mutein molecule streptavidin. In specific embodiments of any of the methods provided herein, the incubation of the second plurality of streptavidin or streptavidin mutein molecules with the thiolating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to thiolating agent between 1:50 and 1:500, inclusive. In some embodiments of any of the methods provided herein, the incubation of the second plurality of streptavidin or streptavidin mutein molecules with the thiolating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to activating reagent of or about 1:100. In some embodiments of any of the methods provided herein, the thiolating agent is or contains 2-iminothiolane.

In specific embodiments of any of the methods provided herein, a second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated at a pH of 7.0 or greater, optionally between 7.0 and 8.0, inclusive. In some embodiments of any of the methods provided herein, a second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated at a pH of approximately 7.7. In some embodiments of any of the methods provided herein, the incubation of the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent is started in the presence of a buffer with a pH of 8.0 or greater, optionally between 8.0 and 9.0, inclusive. In specific embodiments of any of the methods provided herein, the incubation of the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent is started in the presence of a buffer at pH or about 8.5. In some embodiments of any of the methods provided herein, the buffer contains a borate. In some embodiments of any of the methods provided herein, the buffer contains 10 mM-200 mM borate or 50 mM-100 mM borate, always inclusive, optionally about 100 mM borate.

In specific embodiments of any of the methods provided herein, the second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated at a temperature between 4°C and 39°C. In some embodiments of any of the methods provided herein, a second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated at room temperature, optionally between 20°C and 25°C, optionally at or about 23°C, or at or about at 24°C. In some embodiments of any of the methods provided herein, a second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated for 15 minutes to 2 hours, or 15 minutes to 1.5 hours. In specific embodiments of any of the methods provided herein, the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated for 15 minutes to 2 hours, or 25 minutes to 1 hour, always inclusive. In some embodiments of any of the methods provided herein, the second plurality of streptavidin and thiolating agent molecules are incubated for or about 1 hour.

In some embodiments of any of the methods provided herein, a second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated for or about 25 minutes. In specific embodiments of any of the methods provided herein, the molar ratio of the plurality of activated streptavidin or streptavidin mutein molecules to the plurality of thiolated streptavidin or streptavidin mutein molecules during incubation is X:1, where X is the number of lysine residues available for thiolation , per streptavidin or streptavidin mutein molecule. In some embodiments of any of the methods provided herein, the molar ratio is from 1:1 to 8:1, or from 2:1 to 6:1, optionally at or about 4:1. In some embodiments, the mole ratio is from 1:1 to 1:8, or from 1:2 to 1:6, optionally equal to or approximately equal to 1:4.

In some embodiments of any of the methods provided herein, a plurality of activated streptavidin or streptavidin mutein molecules and a plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a pH between 6.8 and 7.5, inclusive. In specific embodiments of any of the methods provided herein, a plurality of activated streptavidin or streptavidin mutein molecules and a plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a pH between 7.0 and 7.4, inclusive. In some embodiments of any of the methods provided herein, a plurality of activated streptavidin or streptavidin mutein molecules and a plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at pH or about 7.2.

In some embodiments of any of the methods provided herein, a plurality of activated streptavidin or streptavidin mutein molecules and a plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a temperature between 4°C and 39°C, inclusive. In specific embodiments of any of the methods provided herein, the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at room temperature, optionally between 20°C and 25°C, inclusive, optionally at or about 23°C or at or approximately at 24°C. In some embodiments of any of the methods provided herein, a plurality of activated streptavidin molecules and a plurality of thiolated streptavidin molecules are incubated for 15 minutes to 6 hours or 30 minutes to 2 hours, always inclusive. In some embodiments of any of the methods provided herein, a plurality of activated streptavidin molecules and a plurality of thiolated streptavidin molecules are incubated for 45 minutes to 1.5 hours, inclusive, optionally within or about 1 hour. In specific embodiments of any of the methods provided herein, the incubation of the activated streptavidin or streptavidin mutein molecules and the thiolated streptavidin or streptavidin mutein molecules is terminated by contacting the molecules with N-ethylmaleimide (NEM).

In some embodiments of any of the methods provided herein, at least part of the incubation of the first plurality of streptavidin molecules or streptavidin mutein with an activating agent and at least part of the incubation of the second plurality of streptavidin molecules or streptavidin mutein with a thiolating agent are performed separately at the same time. same time. In some embodiments of any of the methods provided herein, the incubation of the first plurality of streptavidin or streptavidin mutein molecules with an activating agent and the incubation of the second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent are carried out for essentially the same period of time and/or perform essentially at the same time. In specific embodiments of any of the methods provided herein, prior to incubation of the thiolated streptavidin or streptavidin mutein molecules and the activated streptavidin or streptavidin mutein molecules, the method comprises (i) removing an activating agent from the composition comprising the activated streptavidin or streptavidin mutein molecules; and/or (ii) removing the thiolating agent from the composition containing the thiolated streptavidin or streptavidin mutein molecules.

In some embodiments of any of the methods provided herein, wherein the incubation of the activated streptavidin or streptavidin mutein plurality and the thiolated streptavidin or streptavidin mutein plurality is started within 15 minutes of the end of incubation of the second streptavidin plurality of molecules with the thiolating agent and/or after removing the thiolating agent from the composition containing the thiolated streptavidin or streptavidin mutein molecules.

Provided herein are methods for preparing particulate oligomeric reagents comprising: incubating a first plurality of streptavidin or streptavidin mutein molecules with succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH) for or about 1 hour at a pH of or about equal to 7.2, thus creating a plurality of activated streptavidin or streptavidin mutein molecules containing a maleimide thiol-reactive functional group; incubating a second plurality of streptavidin or streptavidin mutein molecules with 2-iminothiolane for or about 1 hour at a pH between 7.5 and 8.5, inclusive, thereby creating a plurality of thiolated streptavidin molecules containing one or more thiol functional groups; and incubating the plurality of activated streptavidin molecules or streptavidin mutein with the plurality of thiolated streptavidin molecules for or about 1 hour at a pH of or about 7.2, thereby obtaining a composition containing particulate oligomeric reagents; wherein the incubation of the plurality of activated streptavidin molecules with the plurality of thiolated streptavidin molecules is started within 10 minutes after the incubation of the second plurality of streptavidin molecules with 2-iminothiolane ends.

In some embodiments of any of the methods provided herein, the method further comprises contacting the particulate oligomeric reactants with a stabilizing agent. In specific embodiments of any of the methods provided herein, the stabilizing agent reduces the amount of N-substituted iminothiolane present on the lysine residues of the particulate oligomeric reagents. In some embodiments of any of the methods provided herein, the stabilizing agent reduces the amount of N-substituted iminothiolane present on the lysine residues of the particulate oligomeric reagents by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In some embodiments of any of the methods provided herein, the stabilizing agent contains hydroxylamine.

In specific embodiments of any of the methods provided herein, the particulate oligomeric reagents have a radius of less than 150 nm. Some embodiments of any of the methods provided herein further include filter sterilization of particulate oligomeric reagents. In some embodiments of any of the methods provided herein, particulate oligomeric reagents are separated from monomeric or smaller oligomeric streptavidin or streptavidin mutein molecules using size exclusion chromatography. In specific embodiments of any of the methods provided herein, the exclusion limit is greater than or approximately greater than 100 kDa, 500 kDa, 750 kDa, 1000 kDa, or 2000 kDa. In some embodiments of any of the methods provided herein, the exclusion limit is from or about 500 kDa to 1000 kDa. In some embodiments of any of the methods provided herein, the exclusion limit is, or approximately, 750 kDa.

Certain embodiments of any of the methods provided herein include collecting one or more free volume fractions, thereby separating particulate oligomeric reactants from monomeric or smaller oligomeric streptavidin or streptavidin mutein molecules. In some embodiments, the methods further comprise storing the particulate oligomeric reactants at a temperature at or below 4°C, at or below -20°C, or at or below -80°C. In some embodiments, the methods further comprise mixing the particulate oligomeric reagents with one or more agents under conditions to reversibly bind the one or more agents to the particulate oligomeric reagents.

Also provided herein are methods for multimerizing one or more agents with a particulate oligomeric reagent, the method comprising mixing the particulate oligomeric reagent prepared by the methods provided herein with one or more agents under conditions to reversibly bind one or more agents with oligomeric particulate reagents. In particular embodiments of any of the methods provided herein, one or more agents comprise a binding partner, wherein the binding partner is capable of binding to one or more binding sites on the particulate oligomeric reagent. In some embodiments of any of the methods provided herein, the binding partner comprises a streptavidin binding peptide.

In some embodiments of any of the methods provided herein, the binding partner comprises a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8) , Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser- His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln- Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

In specific embodiments of any of the methods provided herein, one or more agents bind or are able to bind to a molecule expressed on the surface of a target cell. In some embodiments of any of the methods provided herein, one or more agents comprise an antibody, optionally a Fab. In some embodiments of any of the methods provided herein, one or more agents is a receptor binding agent that binds or is able to bind to a receptor expressed on the surface of a target cell. In specific embodiments of any of the methods provided herein, the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, thereby inducing or modulating a signal in the target cell.

In some embodiments of any of the methods provided herein, the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3. In some embodiments of any of the methods provided herein, the stimulatory agent is a first receptor binding agent, and the method further comprises reversibly binding to an oligomeric particulate reagent of a second receptor binding agent, wherein the second receptor binding agent is capable of specifically bind to a second molecule on the surface of the target cell, and this binding to the second molecule is optionally capable of inducing or modulating a signal in the target cells.

In specific embodiments of any of the methods provided herein, the second receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains an adhesion molecule or a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule. In some embodiments of any of the methods provided herein, the receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains an adhesion molecule or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule, and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo -/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In specific embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor, and the cytokine receptor is selected from IL-2R, IL-1R, IL-15R, IFN-gammaR , TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor, and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1 , CXCR3 and CXCR4.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand that specifically binds to a cytokine or chemokine receptor. In specific embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein the ligand specifically binds IL-2R, IL-1R, IL- 15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or he represents their biologically active fragment.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein the ligand specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof. In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is an adhesion molecule, and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is their biologically active fragment.

In specific embodiments of any of the methods provided herein, one or more agents is a selective agent, wherein the selective agent binds or is able to bind to a selectable marker that is expressed on the surface of the target cell. In some embodiments of any of the methods provided herein, the target cell is an immune cell. In some embodiments of any of the methods provided herein, the target cell is a lymphocyte or an antigen presenting cell. In specific embodiments of any of the methods provided herein, the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell. In some embodiments of any of the methods provided herein, the target cell is a T cell. In some embodiments of any of the methods provided herein, the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

Certain embodiments are directed to a composition containing particulate oligomeric reactants obtained by the method of any embodiment provided herein. Certain embodiments are directed to a composition containing a plurality of particulate oligomeric reagents produced by a method according to any embodiment provided herein. Some embodiments are directed to a finished product containing a particulate oligomeric reagent according to any of the embodiments provided herein, or a composition according to any of the embodiments presented herein.

Provided herein are methods for modulating cells, the method comprising incubating a cell composition comprising target cells in the presence of a particulate oligomeric reagent according to any of the embodiments provided herein, or in the presence of a composition according to any of the embodiments presented in herein, thus modulating target cells. In some embodiments of any of the methods provided herein, modulating target cells includes activating, enriching, and/or expanding target cells.

Provided herein are methods for culturing cells, the method comprising incubating a cell composition comprising target cells in the presence of a particulate oligomeric reagent according to any of the embodiments provided herein, or in the presence of a composition according to any of the embodiments presented in this document. In some embodiments of any of the methods provided herein, the particulate oligomeric reagent contains a reversible bond with one or more agents. In specific embodiments of any of the methods provided herein, one or more agents bind or are able to bind to a molecule expressed on the surface of a target cell. In some embodiments of any of the methods provided herein, one or more agents comprise an antibody, optionally a Fab.

In some embodiments of any of the methods provided herein, one or more agents is a receptor binding agent that binds or is able to bind to a receptor expressed on the surface of a target cell. In specific embodiments of any of the methods provided herein, the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, thereby inducing or modulating a signal in the target cell.

In some embodiments of any of the methods provided herein, the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3. In some embodiments of any of the methods provided herein, the stimulatory agent is a first receptor binding agent, and the method further comprises reversibly binding to an oligomeric particulate reagent of a second receptor binding agent, wherein the second receptor binding agent is capable of specifically bind to a second molecule on the surface of the target cell, and this binding to the second molecule is optionally capable of inducing or modulating a signal in the target cells.

In specific embodiments of any of the methods provided herein, the second receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains an adhesion molecule or a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule. In some embodiments of any of the methods provided herein, the receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains an adhesion molecule or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule, and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo -/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM. In specific embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor, and the cytokine receptor is selected from IL-2R, IL-1R, IL-15R, IFN-gammaR , TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2. In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor, and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1 , CXCR3 and CXCR4.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand that specifically binds to a cytokine or chemokine receptor. In specific embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein the ligand specifically binds IL-2R, IL-1R, IL- 15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2; and/or the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or he represents their biologically active fragment.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein the ligand specifically binds to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof.

In some embodiments of any of the methods provided herein, the receptor binding agent (second receptor binding agent) is an adhesion molecule, and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is their biologically active fragment.

In specific embodiments of any of the methods provided herein, one or more agents is a selective agent, wherein the selective agent binds or is able to bind to a selectable marker that is expressed on the surface of the target cell. In some embodiments of any of the methods provided herein, the target cell is an immune cell. In some embodiments of any of the methods provided herein, the target cell is a lymphocyte or an antigen presenting cell. In specific embodiments of any of the methods provided herein, the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell. In some embodiments of any of the methods provided herein, the target cell is a T cell. In some embodiments of any of the methods provided herein, the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO. In some embodiments, target cells include blood cells, leukocytes, lymphocytes, B cells, a population of B cells, T cells, a population of T cells, NK cells, dendritic cells, and/or macrophages. In specific embodiments of any of the methods provided herein, the target cells express the recombinant receptor. In some embodiments of any of the methods provided herein, the target cells express a recombinant T cell receptor and/or a chimeric antigen receptor (CAR).

In some embodiments of any of the methods provided herein, the target cells express a CAR that binds to an antigen associated with a disease and/or cancer. In specific embodiments of any of the methods provided herein, the antigen is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer -testicular antigen, cancer-testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), cancer-embryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III (EGFR vIII) mutation, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin-B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc- receptor-like protein 5 (FCRL5; also known as Fc receptor homologue 5 or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folic acid receptor alpha, embryonic acetylcholine receptor, GD2 ganglioside, O-acetylated GD2 (OGD2), GD3 ganglioside, G-protein coupled glycoprotein 100 (gp100) 5D receptor (GPCR5D), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, high molecular weight human melanoma-associated antigen (HMW-MAA), viral surface antigen hepatitis B, human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2 (HLA-A2), IL-22 alpha receptor (IL-22Ra), IL-13 alpha receptor 2 (IL-13Ra2), receptor, kinase insertion domain (kdr), kappa light chain, cell adhesion molecule L1 (L1CAM), CE7 epitope L1-CAM, leucine-rich repeat family A member 8 (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1 , MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegavirus (CMV), mucin 1 (MUC1), MUC16, ligan natural killer group D 2 members (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen gland (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor gene (WT-1), pathogen-specific or universal label-associated antigen and/or biotinylated molecules and/or molecules expressed by HIV, HCV , HBV or other pathogens.

Some embodiments of any of the methods provided herein further include disrupting the reversible binding between one or more agents and the particulate oligomeric reagent. In some embodiments of any of the methods provided herein, said disruption comprises administering to the target cells a composition containing a substance capable of flipping the bond between one or more agents and the particulate oligomeric reagent. In specific embodiments of any of the methods provided herein, the substance is a free binding partner and/or competitive agent.

In some embodiments of any of the methods provided herein, said gap terminates or attenuates the signal induced or modulated by one or more agents in target cells, optionally T cells. In some embodiments of any of the methods provided herein, the substance contains a streptavidin-binding peptide, biotin or a biologically active fragment, optionally D-biotin, or a biotin analog (or biologically active fragment).

In specific embodiments of any of the methods provided herein, the substance is a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His -Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe -Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19) In some embodiments of any of the methods presented in herein, the break is carried out within 5 days after the start of said incubation.

Brief description of drawings

In FIG. 1 shows the levels of thiol functionality coupled to streptavidin mutein tetramers during incubation of an exemplary streptavidin mutein STREP-TACTIN® M2 with 2-iminothiolane in the presence of 100 mM borate.

In FIG. 2 is a graph showing the levels of thiol functionality attached to streptavidin mutein tetramers during incubation of an exemplary STREP-TACTIN® M2 streptavidin mutein with 2-iminothiolane in 25 mM borate buffer at pH 8.3 or 8.5.

In FIG. 3 shows the SEC elution profiles of the streptavidin mutein incubated with 2-iminothiolane. Elution peaks for molecular weight standards are shown as a solid line for molecular weights of 158,000 Da, 44,000 Da, 17,000 Da, or 1350 Da. The elution profile of the non-thiolated exemplary streptavidin mutein STREP-TACTIN® M2 is shown as a dotted line. The remaining elution profiles depict the elution profiles of various thiolated STREP-TACTIN® M2 streptavidin muteins that were incubated in the presence of 25 mM borate buffer at pH 8.3 or pH 8.5 for either 10 minutes, or 50 minutes, or 390 minutes. Specifically shown are the elution profiles after incubation of exemplary streptavidin mutein STREP-TACTIN® M2 with 2-iminothiolane in the presence of 25 mM borate buffer at pH 8.3 for 10 minutes, 25 mM borate buffer at pH 8.3 for 50 minutes, 25 mM borate buffer at pH 8.3 for 390 minutes or 25 mM borate buffer at pH 8.5 for 10 minutes, 25 mM borate buffer at pH 8.5 for 50 minutes or 25 mM borate buffer at pH 8.5 in within 390 minutes.

In FIG. 4 shows the concentration of thiol functionality (SH content) after incubation of exemplary streptavidin mutein STREP-TACTIN® M2 with 2-iminothiolane for 1 hour or 3 hours with 25 mM borate buffer at pH 8.3, 8.5 and 8.7.

In FIG. 5 shows the loss of thiol functionality (SH content) of an exemplary streptavidin mutein STREP-TACTIN® M2 at different time points after incubation with 2-iminothiolane and gel filtration with PD10 columns.

In FIG. 6A and 6B show the results of a metabolic analysis of WST T cells from three different donors incubated with anti-CD3/anti-CD28 antibodies multimerized on different lots of oligomeric reagents consisting of the exemplary streptavidin mutein STREP-TACTIN® M2. In FIG. 6A shows the metabolic activity of WST for all lots tested (pooled) compared to reference lots containing anti-CD3/anti-CD28 antibodies multimerized on an oligomeric backbone with a mean hydrodynamic radius of 36 nm or 101 nm. In FIG. 6B shows the average metabolic activity of WST between T cells from different donors for individual batches tested and reference reagents.

In FIG. 7 which includes FIG. 7A-7E are schematic representations of exemplary embodiments.

In FIG. 7A is a schematic representation of a reagent (or a representative portion thereof), such as streptavidin oligomeric reagent or streptavidin mutein, with multiple binding sites for reversible binding to agents. In this case, a reagent is shown that can reversibly bind to two agents, each of which is able to specifically bind to a molecule on a cell. The reagent has a plurality of binding sites, including a plurality of binding sites, Z1, each of which is capable of reversibly binding to agents. Each of the first and second agents, which in some cases may be the same, in the shown schematic representation contain at least one C1 binding partner. The C1 binding partner binds reversibly to the Z1 binding site. Each of the first and second agents also contains a binding site, B2, which can specifically bind to a molecule on the cell surface, which in some cases may be on the same cell. In this case, it is shown that the first and second agents bind specifically to molecules on the same cell.

In FIG. 7B is a schematic representation of a reagent, such as a streptavidin oligomeric reagent or a streptavidin mutein, with a plurality of binding sites capable of reversibly binding to a first and second agent, each of the agents being able to specifically bind to a molecule on the first and second cell, respectively. The reagent has a plurality of Z1 binding sites, each of which is capable of reversibly binding to the agent. Each of the first and second agents, which may be the same in some cases, contains a C1 binding partner that reversibly binds to the Z1 binding site. Each of the first and second agents contains a B2 binding site that can specifically bind to a molecule on the surface of a cell, which in some cases can be on the same cell or on a different cell. In this case, the first agent is associated with a molecule on the surface of the first cell, and the second agent is associated with a molecule on the surface of the second cell.

In FIG. 7C shows a reagent, such as streptavidin oligomeric reagent or streptavidin mutein, capable of reversibly binding to the first and second agents, each of the agents being able to specifically bind to a molecule on the first and second cell, respectively. The reagent has a plurality of Z1 and Z2 binding sites, which may be the same or different, each capable of reversibly binding to one or both agents. The first agent contains a C1 binding partner that binds reversibly to Z1; the second agent contains a C2 binding partner that can reversibly bind to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains a B1 binding site that can specifically bind to a molecule on the cell surface, and the second agent contains at least one B3 binding site that can specifically bind to a molecule on the cell surface. The B1 and B3 binding sites in some cases bind to two different molecules on the cell surface, or to different epitopes on the same molecule, or to the same or different molecules on the surface of different cells. In this case, it is shown that the first agent is associated via B1 with a molecule on the surface of the first cell, and the second agent is associated with a molecule on the surface of the second cell.

In FIG. 7D shows a reagent, such as a streptavidin oligomeric reagent or a streptavidin mutein, capable of reversibly binding to a first and second agent, such as selective agents, each of which is capable of specifically binding to a molecule on a cell. The reagent has a plurality of binding sites, including Z1 and Z2, which may be the same or different, each of which is capable of reversibly binding to the agent. The first agent contains a C1 binding partner that can specifically bind to the Z1 binding site, and the second agent contains at least one C2 binding partner that can specifically bind to the Z2 binding site. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains a B1 binding site that can specifically bind to a molecule on the cell surface, and the second agent contains a B3 binding site that can specifically bind to a molecule on the cell surface. In some embodiments, the first agent and the second agent may be a selective agent. Binding sites B1 and B3 can bind the same or different molecules (eg, a receptor) on a cell surface, the same or different epitopes on a molecule, or the same or different molecules on the surface of different cells. In this case, the first agent is associated with the first molecule on the surface of the cell, and the second agent is associated with the second molecule on the surface of the same cell.

In FIG. 7E shows a reagent, such as a streptavidin oligomeric reagent or a streptavidin mutein, reversibly associated with a first and a second agent, each of which is capable of specifically binding to a molecule on a cell. The reagent has a plurality of binding sites, including Z1 and Z2, which may be the same or different, each of which is capable of reversibly binding to the agent. The first agent contains a C1 binding partner that can reversibly bind to Z1 of the reagent, and the second agent contains a C2 binding partner that can reversibly bind to Z2. In some cases, C1 and C2 are different. In some cases, C1 and C2 are the same or substantially the same. The first agent contains at least one B2 binding site that can specifically bind to a molecule on the cell surface, and the second agent contains at least one B4 binding site that can specifically bind to a molecule on the cell surface. In some embodiments, the first agent and the second agent may be stimulant agents. The B2 and B4 binding sites can bind the same or different molecules on a cell surface, the same or different epitopes on a molecule, or the same or different molecules on the surface of different cells. In this case, the first agent is associated with the first molecule on the surface of the cell, and the second agent is associated with the second molecule on the surface of the same cell.

In FIG. 8 which includes FIG. 8A-8E are schematic representations of the exemplary embodiments shown in FIG. 7A-7E, respectively, except that depicted reagents such as streptavidin oligomeric reagent or streptavidin mutein are shown immobilized on a carrier such as a stationary phase.

In FIG. 9 is a schematic representation of exemplary embodiments in which oligomeric reagents such as streptavidin oligomeric reagent or streptavidin mutein are used to multimerize stimulatory agents and the resulting complexes are incubated with cells to deliver signals to cells, followed by reversal of binding. Panel A shows oligomeric reagent 1, which is shown to be unbound to any carrier and flexible. Stimulating agents 2, which are shown here as Fab fragments and are able to specifically bind to a molecule on the cell surface, are combined with a reagent. The agents contain a binding partner (eg, binding partner C) that is capable of reversibly binding to a binding site (eg, binding site Z) on the reagent, multimerizing the agents. Panel B shows a binding partner reversibly binding to a binding site on a reagent. Cells 3 are added to the system. Panel C shows multimerized agents (Fab fragments) specifically binding to molecules 4 on the surface of cell 3. Panel C depicts agents that are stimulatory receptor binding agents (e.g., the first receptor binding agent and/or second receptor-binding agent) that can induce or modulate a signal in a cell when the agent binds to a molecule on the cell. As shown in panel D, substance 5, such as a competitive reagent (e.g., biotin), is added to the composition, which may be a substance that exhibits a higher binding affinity for the binding site on the reagent than for the binding partner on the agent, thus disrupting reversible binding between reactant 1 and agent 2. In some cases, an agent, such as a Fab fragment, can also dissociate from its interaction with molecule 4 on cell 3. In some cases, this can disrupt, attenuate, and/or stop cell signaling.

In FIG. 10 is a schematic representation of exemplary embodiments of a reversible system including oligomeric reagents, such as streptavidin oligomeric reagent or streptavidin mutein, coupled to a carrier, such as a solid carrier or a surface containing a stationary phase. Panel A shows carrier 6 containing reagent 1. Agents 2 are added to the system, such as Fab fragments that are able to specifically bind to a molecule on the cell surface. Agents 2, such as Fab fragments, contain a binding partner (eg, binding partner C) that is capable of reversibly binding to a binding site (eg, binding site Z) on the reagent. Panel B shows a binding partner reversibly binding to a binding site on a reagent. Cells 3 are added to the system. Panel C shows 2 agents, such as Fab fragments that bind to molecules 4 on the surface of cell 3. In some embodiments, the scFvs contain a receptor binding agent or a selective agent. In some embodiments, the agents, such as Fab fragments, may be a receptor binding agent or a selective agent. Panel C shows an exemplary receptor binding agent or agents (e.g., a first receptor binding agent and/or a second receptor binding agent) that can induce or modulate a signal in a cell upon binding of an agent, e.g., a Fab fragment, to a molecule on cell. Substance 5 is added, such as a competitive reagent (e.g., biotin), which may be a substance that exhibits a higher binding affinity for a binding site on the reagent than for a binding partner on the agent, for example, a Fab fragment, thus disrupting the binding between reagent and agent. Panel D shows the disruption of binding between agent 2, eg a Fab fragment, and the reagent, thus leading to dissociation of the reagent from the agent, and thus the cell. In some cases, an agent, such as a Fab fragment, may also dissociate from its interaction with molecule 4 on cell 3. In some cases, this may disrupt, attenuate and/or stop signaling in the cell.

In FIG. 11 is a plot of size changes measured by dynamic light scattering of streptavidin mutein oligomeric reagents at different time points after storage at -80°C, 4°C or 37°C.

In FIG. 12A and B are plots of total cell count (FIG. 12A) and percentage viable cells (FIG. 12B) over time during incubation with different individual lots of anti-CD3/anti-CD28 Fab-conjugated streptavidin mutein oligomeric reagents during an exemplary design process. to create compositions of T cells containing T cells expressing a chimeric antigen receptor (CAR).

In FIG. 13 is a graph showing the percentages of total CAR+, CD4+CAR+, and CD8+CAR+ cells, as well as the percentage of CAR+CD4+ T cells among CD4+ T cells and the percentage of CAR+CD8+ T cells among CD8+ T cells of the compositions T cells incubated with different separate batches of anti-CD3/anti-CD28 Fab-conjugated streptavidin mutein oligomeric reagents during an exemplary design process to generate CAR T cell compositions.

In FIG. 14 is a graph showing the percentage of cells positive for residual Fab staining (upper panel) or residual streptavidin mutein (lower panel) among cell compositions that were incubated with different individual batches of Fab-conjugated streptavidin mutein oligomeric reagents.

In FIG. 15 is a graph depicting the cytotoxic activity of T cell compositions containing CAR T cells that were generated using an exemplary design method that included incubation with different separate batches of anti-CD3/anti-CD28 Fab-conjugated streptavidin mutein oligomeric reagents.

Detailed description

Unless otherwise indicated, all art terms, designations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as generally understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some instances, terms with conventional meanings are defined herein for clarity and/or ease of reference, and the inclusion herein of such definitions should not necessarily be construed as representing a material difference from what is commonly understood in the art.

All publications, including patent documents, scientific articles and databases referred to in this application, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If the definition herein conflicts with or is otherwise inconsistent with the definition set forth in patents, applications, published applications, and other publications that are incorporated herein by reference, the definition set forth herein takes precedence over the definition that is incorporated herein by reference. links.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the scope of the invention.

I. Overview

This document provides methods for the manufacture, preparation and/or creation of particulate oligomeric reagents. In some embodiments, the methods provided herein are used to oligomerize molecules into oligomeric particulate reactants in the range of 1 million Da to 100 million Da, 1 million Da to 1 billion Da, 1 million Da to 10 billion Da, and /or from 1 million Yes to 100 billion Yes. In certain embodiments, it is contemplated that the size distribution of the particulate oligomeric reactants produced by the present methods is affected by varying individual reaction conditions at one or more different steps. Thus, in some embodiments, conditions such as timing, concentrations and molar ratios of the reactants and the pH of the solutions are precisely controlled and kept constant. In some embodiments, the implementation of the methods presented herein, used to obtain oligomeric reagents in the form of particles with stable sizes between batches or batches. In some embodiments, the conditions of one or more of the steps or steps of the methods provided herein can be adjusted to make, obtain, or create oligomeric particulate reactants with one or more different desired sizes.

In some embodiments, the methods provided herein crosslink molecules, e.g., streptavidin or streptavidin mutein tetramers, by reacting a plurality of molecules having attached thiol functional groups (also called thiolated molecules, for example, thiolated streptavidin or streptavidin mutein molecules) with a plurality of molecules having a thiol-reactive functional group attached (for example, activated molecules, for example, streptavidin molecules or streptavidin mutein), such as a maleimide functional group.

In certain embodiments, it is contemplated that multimerization reagents and/or oligomerization reagents in the form of particles of one or more specific particle sizes may be particularly effective for use in cell modulation. For example, in some embodiments, the implementation of oligomeric reagents in the form of particles of a certain size when reversibly associated with one or more stimulating agents are particularly effective in expanding, activating and/or enriching a population of cells. As found herein, oligomeric reagents in the form of particles of a particularly large size, for example, having an average radius, for example, a hydrodynamic radius, greater than 32 nm, and usually an average radius greater than 60 nm, for example, an average radius greater than or approximately greater than 90 nm , 95 nm or 100 nm, which are reversibly associated with stimulating agents, activate cells to a greater extent than smaller oligomeric particles, such as FIG. 6. Thus, in some embodiments, provided herein are particulate oligomeric reactants with defined sizes and size distributions, and methods for stably producing particulate oligomeric reactants with desired sizes and size distributions.

In some embodiments, the present methods for producing particulate oligomeric reactants result in reduced variability and generally consistent production of larger oligomeric reactants while minimizing the size distribution of the oligomers in the composition. In some embodiments, reactive thiol groups for the oligomerization reaction are added to the molecules by activating iminothiolane amines present at the lysine residues and at the N-terminus of the molecules. In some embodiments, the timing of the thiolation reaction and the time between the end of the thiolation reaction and the start of the oligomerization reaction are kept constant from reaction to reaction in some cases because free thiols can be lost due to isomerization, i. Formation of N-substituted iminothiolane. In some aspects, the timing should minimize the loss of thiols and the creation of the N-substituted form of iminothiolane, which in some cases is a hidden source of SH function in re-isomerization, which can lead to postsynthetic growth of oligomers. In some embodiments, controlling the availability of thiol groups for reaction with maleimide-containing molecules, and minimizing the accumulation of N-substituted iminothiolane, is a parameter that can influence oligomer size consistency.

In addition, in some embodiments, the implementation of the kinetics of activation of the thiol and also other chemical reactions in some cases may depend on pH. In some embodiments, the pH of one or more reaction buffers (activation and binding buffers), and in particular the pH of the thiolating agent buffer, is within a predetermined range or level, and is typically accurately measured and controlled. In addition, in some embodiments, the stoichiometry between the thiolating and activating agents and molecules is controlled with high precision by adjusting the concentrations within small tolerances (± 2%) to obtain reproducible average multimer sizes.

Also provided herein are such oligomeric reagents as described herein. In some embodiments, one or more agents may be reversibly or irreversibly associated with oligomeric reagents, which in some cases are a multimerization agent, wherein one or more agents are multimerized on the particulate oligomeric reagents. Oligomeric reagents having one or more agents associated with them, such as multimerization agents, can be used in methods involving culturing or incubating with target cells, including primary cells such as T cells.

In some aspects, provided herein are particulate oligomeric reagents that are linked or reversibly linked (eg, multimerized) with one or more agents, such as receptor binding reagents and/or stimulatory reagents. In specific embodiments, the provided oligomeric particles are composed of oligomerized molecules, eg, cross-linked streptavidin muteins, and are associated with stimulatory and/or receptor binding agents that bind and/or are able to bind to the cell surface. In some aspects, the agents are or comprise an anti-CD3 and/or anti-CD28 antibody, or antigen-binding fragments thereof, having a binding partner, for example, a streptavidin-binding peptide such as Strep-tagII. In specific embodiments, the oligomerized particulate reagents have a radius between 50 nm and 150 nm, 75 nm and 125 nm, 80 nm and 115 nm, or a radius equal to or approximately equal to 80 nm, 85 nm, 90 nm, 95 nm, 100 nm , 105 nm, 110 nm, 115 nm ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1% or ±0.1%. In some aspects, a composition is provided comprising a plurality of particulate oligomeric reactants, such as reactants bonded or reversibly bonded (e.g., multimerized) to one or more agents, wherein the mean radius of the particulate oligomeric reactants in the plurality is between 50 nm and 150 nm, 75 nm and 125 nm, 80 nm and 115 nm, or radius equal to or approximately equal to 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm ±25%, ±20%, ±15%, ±10%, ±5%, ±2%, ±1%, or ±0.1% In some aspects, particulate oligomerized reagents are particularly suitable for cell selection and/or stimulation, for example, by binding a selective agent or a stimulating agent, respectively, with a cell surface molecule on target cells. In some cases, the presence or addition of a competitive reagent results in dissociation between the particulate oligomeric reagent and agents, e.g., receptor binding reagents, which in some cases can rapidly terminate, terminate, or disrupt cell stimulation with the particulate oligomeric reagents.

This document provides a method for propagating a composition of target cells, such as T cells, using the presented oligomeric reagents. In some embodiments, the methods refer to reversible reagent systems capable of binding to molecules on the surface of target cells, such as a receptor-binding molecule, thereby providing a signal to the cells, which in some cases may be a primary activation signal. In some embodiments, the particulate oligomeric reagents used in the methods are multimerization reagents and/or particulate oligomeric reagents having one or more agents bound thereon, e.g., a first agent, a second agent, etc., which provides a signal to the cells, such as a primary activation signal and/or an additional or costimulatory signal. In some embodiments, the implementation of the primary activation signal by itself may be sufficient to activate the reproduction/proliferation of cells. This first agent may be either reversibly or also irreversibly associated with the multimerization reagent and/or particulate oligomeric reagents. The multimerization reagent and/or particulate oligomeric reagents may also have a second agent associated with them that stimulates a helper molecule on the cell surface. The second agent, when bound to a helper molecule on the cell surface, can thus stimulate the proliferation of activated cells. Also, this second agent can be either reversibly or also irreversibly linked to the multimerization reagent and/or the particulate oligomeric reagent. The multimerization reagent and/or the particulate oligomerization reagent may be either immobilized on a solid support or soluble. In one aspect, the method disclosed herein is a cell population serial expansion in which a complete population of lymphocytes is stimulated/expanded, then reagents necessary for expansion are removed by chromatography on a suitable stationary phase. In some embodiments, the expanded/stimulated cells, which are cultured cells, are optionally transfected with, for example, a T cell receptor or a chimeric antigen receptor (CAR), and in some aspects can be subjected to a second expansion stimulated with another stimulatory molecule that binds to introduced T-cell receptor or chimeric antigen receptor.

This document provides a method for propagating a composition of target cells, such as T cells. In some embodiments, the methods involve reversible reagent systems capable of binding to molecules on the surface of target cells, such as a receptor-binding molecule, thereby providing a signal to the cells, which in some cases may be a primary activation signal. In some embodiments, the methods employ reagents such as particulate oligomeric reagents, which may be multimerization reagents, and/or a particulate oligomeric reagent having one or more agents bound thereon, e.g., a first agent, a second agent, etc. .d. Which provides a signal to the cells, such as a primary activation signal and/or an additional or costimulatory signal. In some embodiments, the implementation of the primary activation signal by itself may be sufficient to activate the reproduction/proliferation of cells. This first agent may be either reversibly or also irreversibly associated with the multimerization reagent and/or the particulate oligomeric reagent. The multimerization reagent and/or the particulate oligomeric reagent may also have a second agent associated therewith that stimulates an accessory molecule on the cell surface. The second agent, when bound to a helper molecule on the cell surface, can thus stimulate the proliferation of activated cells. Also, this second agent can be either reversibly or also irreversibly linked to the multimerization reagent and/or the particulate oligomeric reagent. The multimerization agent may be either immobilized on a solid support or soluble. In one aspect, the method disclosed herein is a cell population serial expansion in which a complete population of lymphocytes is stimulated/expanded, then reagents necessary for expansion are removed by chromatography on a suitable stationary phase. In some embodiments, the expanded/stimulated cells, which are cultured cells, are optionally transfected with, for example, a T cell receptor or a chimeric antigen receptor (CAR), and in some aspects can be subjected to a second expansion stimulated with another stimulatory molecule that binds to introduced T-cell receptor or chimeric antigen receptor.

Methods for propagating a population of T cells in vitro in the absence of exogenous growth factors or at low levels of exogenous growth factors are known in the art (see, for example, US Pat. No. 6,352,694 B1 and EP 0 700 430 B1). In general, such methods involve solid phase surfaces larger than 1 μm in diameter, on which various binding agents (eg, anti-CD3 antibody and/or anti-CD28 antibody) are immobilized. For example, Dynabeads® CD3/CD28 T cell expansion reagents (Invitrogen) are commercially available, which are homogeneous, 4.5 µm diameter, superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of affinity purified monoclonal antibodies against CD3 molecules and Cell surface CD28 on human T cells. However, in some cases, such magnetic beads, for example, are difficult to integrate into a cell expansion method under the conditions required for clinical trials or therapeutic purposes, since it is necessary to ensure that these magnetic beads are completely removed before administering expanded T cells to a patient.

In some embodiments, the implementation of the methods presented in this document, solve these problems. In some aspects, the provided reagents are reversible so that the stimulating agents can be removed from the cell composition. Also, in some aspects, the reagent, eg, the multimerization reagent or the particulate oligomeric reagent, to which the stimulating agents are associated, is not immobilized on the support, eg, not immobilized on a solid support or surface. Thus, in some aspects, the reagent, for example, the multimerization reagent and/or the particulate oligomeric reagent, is flexible rather than rigid. In some embodiments, the implementation of the reagent may adapt or match the surface of the cell. In some embodiments, the reagent may be immobilized on a carrier, such as a solid carrier, including a stationary phase. In some embodiments, such methods can be used in conjunction with selection methods using similar selective agents, in which one or more target cells can be selected and simultaneously or sequentially exposed to stimulating agents. Therefore, in some aspects, the stimulation of individual cells or subgroups of cells can be shifted by selection and isolation along with stimulation.

In some embodiments, the methods provided include culturing, e.g., contacting the cell composition with a reagent, e.g., a multimerization reagent or a particulate oligomeric reagent, to which one or more receptor binding agents (e.g., stimulatory agents) are bound (see, for example, Fig. 10A and 10B). In some embodiments, after contacting the cell composition with a multimerization reagent and/or particulate oligomeric reagent with one or more coupled receptor-binding agents, and typically incubating the cell population with the multimerization reagent and/or particulate oligomeric reagent with one or more associated receptor-binding agents, the cell population forms complexes/associates with the multimerization agent through the first agent. Other cell populations contained in the original sample that do not have a particular cell surface molecule do not bind to the multimerization reagent and/or the particulate oligomeric reagent with one or more associated receptor binding agents. In this regard, it should be noted that a population of cells typically has multiple copies of a cell surface molecule on its surface, and binding of these multiple copies is usually required for stimulation or activation.

Thus, a multimerization agent typically provides more than one binding site, for example, Z1, in which in some cases a plurality of agents can be reversibly linked, for example, by binding a binding partner, for example, C1, one or more agents to one or more sites binding, for example, Z1. In some such aspects, the first agent, the second agent, and/or other agents are provided at a sufficient density in the cell population. In this regard, it should be noted that the multimerization agent itself may have multiple binding sites, for example, Z1, for example, the streptavidin mutein (which is a homotetramer) in its native state has four such binding sites, for example, Z1, and may additionally be oligomerized. In some cases, the reagent may have only one binding site, for example, Z1, for reversible binding of the binding partner, for example, C1. An example of this is the multimeric calmodulin. Calmodulin itself has only one binding site for calmodulin-binding peptides. However, calmodulin can be biotinylated and then reacted with streptavidin oligomers (see also below), thus providing a multimerization reagent in which many calmodulin molecules are present at high density on a "scaffold", thus providing a multimeric calmodulin.

In some embodiments, after incubation or other appropriate time at which stimulation is to be interrupted, binding between a binding partner, also referred to herein as binding partner C, e.g. C1, of a reversibly bound agent, and a binding site Z, e.g. the multimerization reagent and/or the particulate oligomeric reagent is disrupted by breaking the corresponding reversible bond. In some cases, disruption can be achieved by adding a competitor to an incubation/reaction mixture containing a population of cells associated with the multimerization reagent and/or particulate oligomeric reagents. For competitive cleavage (which can be understood as competitive elution) of the reversible bond between the binding partner C, e.g. C1, of the reversibly bound agent and the Z binding site, e.g. Z1, multimerization reagent and/or particulate oligomeric reagents, incubation mixture/ the cell population can be contacted with a free first binding partner C, eg C1, or an analogue of said first binding partner C, which is capable of breaking the bond between the first binding partner and the binding site Z, eg Z1. In an example of a binding partner C, eg C1, which is a streptavidin binding peptide that binds to the streptavidin biotin binding site, the first free partner may be the corresponding free streptavidin binding peptide or analog that binds competitively. Such an analog can be, for example, biotin or a derivative or analog of biotin, such as dethiobiotin.

In some embodiments, the addition of a free partner or equivalent results in displacement of the binding partner C, e.g. such an agent from a multimerization reagent and/or a particulate oligomeric reagent. This displacement of the agent, in turn, results in the dissociation of the first agent from the cell surface molecule, in particular if the binding affinity of the bond between the first agent and the cell surface receptor has a dissociation constant (K _d ) in the range from 10 ^-2 M to 10 ^-13 M and thus is also reversible. Due to such dissociation, in some aspects, stimulation of the cell population is also terminated.

In some embodiments, the binding affinity of antibody molecules to their antigen, including, for example, a cell surface receptor molecule, is typically in the K _d affinity range of 10 ^-7 M to 10 ^-13 M. Thus, conventional monoclonal antibodies can be used as an agent (first or second, receptor-binding, for example, stimulating agent or selective agent). In some embodiments, monoclonal antibodies can also be used in the form of their monovalent antibody fragments, such as Fab fragments or single chain Fv fragments, to avoid any undesirable avidity effects that result in stronger binding.

In some embodiments, due to dissociation of the reversibly bound agent or agents from the cell surface molecule, the present method has the additional advantage that the stimulated cell population does not contain stimulatory agents at the end of the stimulation period. Also in some embodiments, all other reagents used in the method, namely agents (e.g., first or second, receptor binding agents, e.g., stimulatory agents, or selective agents), as well as competitive binding partner reagent C, e.g., C1 , or its analogue can be easily removed from the stimulated cell population by means of a "removal cartridge" (see, for example, described in international patent application WO 2013/124474). In some cases, for example, when the multimerization reagent and/or the particulate oligomeric reagent is immobilized on a solid carrier, such as the surface of a bioreactor or a magnetic bead, it is at the back. Thus, the use of a removal cartridge to remove the free agent and the competitive reagent may involve loading an elution sample (eg, a sample obtained after a reversible bond or bond is broken) onto a second chromatographic column.

In some embodiments, the chromatographic column has a suitable stationary phase that is both an affinity chromatography matrix and at the same time can act as a gel permeation chromatography matrix. In some aspects, the affinity chromatography matrix has an affinity reagent immobilized thereon. In some embodiments, the affinity reagent may be, for example, streptavidin, streptavidin mutein, avidin, avidin mutein, or a mixture thereof. In some embodiments, an agent (e.g., first or second receptor binding agents, e.g., stimulating agents or selective agents), a competitive binding partner C reagent, C1, binds to an affinity reagent, thereby immobilizing on a chromatography matrix. As a result, the eluted sample containing the isolated and expanded population of cells is depleted of the agent (eg, first or second receptor binding agents, eg, stimulatory agents or selective agents) and the competitive reagent. In some embodiments, the cultured composition does not contain any of the reagents that in some aspects are preferred for use in connection with diagnostic applications (eg, additional FACS ^™ sorting) or for any cell-based therapeutic application.

In some embodiments, the ability to remove the reagent and other components from the composition has the added benefit of not being able to avoid any solid support, such as magnetic beads. In some embodiments, this means that there is no risk or minimal risk of contamination of activated T cells with such magnetic beads. In some embodiments, this also means that a method that complies with GMP standards can be more easily created compared to other methods, such as using Dynabeads®, in which additional steps must be taken to ensure that the resulting expanded population of T cells does not contained magnetic balls. In addition, in some embodiments, the use of a soluble multimerization agent greatly facilitates its removal from a population of activated cells (T cells, B cells, or also natural killer cells), since cells can simply be precipitated by centrifugation and supernatant, including soluble the multimerization agent can be discarded. Alternatively, the soluble multimerization agent can be removed from the expanded cell population in a removal cartridge gel permeation chromatography matrix such as described above (eg, in international patent application WO 2013/124474). In some embodiments, since no solid phase (eg, magnetic beads) is present, the present invention also provides an automated closed cell expansion system that can be integrated into known cell expansion systems such as the Xuri Cell Expansion System W25 and WAVE Bioreactor 2/10 System sold by GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) or QUANTUM® Cell Expansion System sold by TerumoBCT Inc. (Lakewood, CO, USA).

In some aspects, the methods provided herein may include the presence of a population of cells that carry at least two specific cell surface molecules. In some embodiments, the first cell surface molecule is involved in the primary activation signal for the cell population, while the second cell surface molecule is an accessory molecule on the cell surface that is involved in providing a stimulus to the cells. In specific embodiments, a population of cells is contacted with a multimerization reagent and/or particulate oligomeric reagents to which a first agent is reversibly or irreversibly associated that provides the cells with a primary activation signal and a second agent that elicits or modulates an additional signal, e.g. , stimulates an accessory molecule on the cell surface. In some embodiments, a population of cells is contacted with a multimerization reagent and/or a particulate oligomeric reagent to which is reversibly associated a first agent that provides the primary activation signal to the cells and a second agent that elicits or modulates an additional signal, e.g., stimulates accessory molecule on the cell surface. The cell population, for example, may be a population of T cells in which the cell surface molecule is a TCR/CD3 complex and the cell surface molecule is an accessory CD28 molecule. In some aspects, stimulation with these other additional molecules can result in an increase in the less differentiated and in some cases long-lived T cell population, such as long-lived memory T cells, compared to conventional stimulation with CD28. In some embodiments, binding of both the TCR/CD3 complex as a primary activation signal and binding of an accessory molecule (eg, CD28 or other accessory molecule) may be required for T cell expansion/proliferation.

In some embodiments, the methods provided herein may also be further combined to include at least one selective agent reversibly linked to the same reagent, e.g., the same multimerization reagent and/or particulate oligomeric reagents, as one or both of the first or second receptor binding agent (eg, a stimulant agent). In some instances, one or more incubation or culture-mediated features, such as promotion of multiplication (proliferation), activation, costimulation, and/or survival, can be enhanced or enhanced in a subset of T cells that can be reversibly selected in the presence of at least one or more selective agents during incubation or cultivation, which also occurs in the presence of one or more stimulating agents. For example, as shown in the examples herein, the expansion rate in a T cell composition in CD8+ cells was selectively increased when such cells were incubated with a multimerized agent to which an anti-CD8 antibody was reversibly linked in addition to the stimulatory agents of an anti-CD3 antibody and an anti-CD8 antibody. CD28. In some embodiments, one or more features due to incubation or culture, for example, stimulation of reproduction (proliferation), activation, costimulation and/or survival, can be increased by at least 1.5 times, at least 2 times, at least at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10-fold or more in the subset of T cells in the culture composition that are positive for the selection marker when incubated in the presence of one or more stimulatory agents and a selection agent that specifically binds to the selection marker, compared to incubation in the presence of one or more alone stimulating agents, but not a selective agent. This bias or selectivity of a cell, such as a T cell, allows the endpoint characteristics of particular subsets or populations of T cells to be controlled. In some embodiments, the implementation of the selective marker can be any selectable marker, which is described in this document. In some embodiments, the selection marker is selected from CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

In some embodiments, the multimerization reagent and/or the particulate oligomeric reagent contains at least one Z binding site, e.g., Z1, for reversibly binding the first agent, and the first agent also contains at least one C binding partner, e.g., C1 , wherein the C binding partner, eg C1, is capable of reversibly or irreversibly binding to the Z binding site, eg Z1, of the multimerization reagent and/or the particulate oligomeric reagent. Thus, the first agent, when brought into contact with or incubated with a multimerization agent, can reversibly bind to the multimerization reagent and/or the particulate oligomeric reagent via a reversible bond formed between the binding partner C, for example, C1, and the binding site Z, for example, Z1. Further, the second agent may comprise a C binding partner, e.g. C2, wherein the C2 binding partner is capable of reversibly binding to the Z binding site, e.g. Z2, respectively, of the multimerization reagent and/or the particulate oligomeric reagent. In some embodiments, the second agent, when contacted or incubated with a multimerization agent, reversibly binds to the multimerization reagent and/or particulate oligomeric reagents via a reversible bond formed between a binding partner C, e.g., C1, and a binding site Z, e.g. , Z2. In some cases, C1 and C2 may be the same or substantially the same and/or contain the same or substantially the same fragment. In some cases, Z1 and Z2 may be the same or substantially the same and/or contain the same or substantially the same fragment.

In some embodiments, the use of fragments that bind to the same binding site of the multimerization agent as C1 and C2 binding partners has the advantage that the same competitive reagent (the first C1 binding partner, as well as a second C2 binding partner) or an analogue thereof for disrupting and in some cases stopping the reproduction of a population of target cells (for example, T cells) and for releasing this population of target cells (for example, T cells) from the multimerization agent.

In some cases, in order to obtain binding agents (for example, first or second receptor binding agents, for example, stimulating agents or selective agents) to have a binding partner C, it is possible to provide a binding partner C, for example, C1 or C2, using the appropriate an expression vector used to recombinantly produce an agent (eg, an antibody fragment), such that binding partner C, eg, C1 or C2, is part of a fusion peptide with the agent at either the N-terminus or the C-terminus. In some embodiments, in the context of an agent that is an antibody or antigen-binding fragment, a C binding partner, such as C1 or C2, may be present at the C-terminus of either the light or heavy chain. Also, this technique for cloning a recombinant protein, eg, the variable domains of an antibody molecule, and recombinantly producing the corresponding protein, eg, an antibody fragment, is well known to those skilled in the art, see, eg, Skerra, A. (1994). In some embodiments, an antibody molecule can be constructed from artificial binding molecules with antibody-like properties against a given target, such as CD3 or CD28, or other complementary or stimulatory agent molecules, as described, for example, using well-known evolutionary techniques such as phage display ( discussed, for example, in Kay, B. K. et al (1996) Phage Display of Peptides and Proteins - A Laboratory Manual, 1st Ed., Academic Press, New York NY Lowman, H. B. (1997) Annu Rev. Biophys Biomol. Struct. 26, 401-424, or Rodi, D. J., and Makowski, L. (1999) Curr. Opin. Biotechnol. 10, 87-93), ribosome display (reviewed in Amstutz, P. et al. (2001) Curr Opin Biotechnol 12, 400-405) or mRNA display as reported in Wilson, D.S. et al. (2001) Proc. Natl. Acad. sci. USA 98, 3750-3755.

II. Reversible Reagent Systems and Related Applications

In some embodiments, methods employ reversible systems wherein at least one agent (e.g., a receptor binding agent or selective agent) capable of binding to a cell surface molecule (cell surface molecule) is bound, e.g., reversibly bound, to reagent. In some instances, the reagent contains a plurality of binding sites capable of binding, for example, reversibly binding to an agent (eg, a receptor binding agent or a selective agent). In some instances, the reagent is a multimerization reagent and/or a particulate oligomeric reagent having at least one agent associated therewith. In some embodiments, at least one agent (e.g., a receptor binding agent or selective agent) contains at least one B binding site that can specifically bind an epitope or region of a molecule, and also contains a binding partner, also referred to herein a binding partner C that specifically binds to at least one Z binding site of the reagent. In some instances, the binding interaction between binding partner C and at least one binding site Z is a non-covalent interaction. In some instances, the binding interaction between binding partner C and at least one binding site Z is a covalent interaction. In some embodiments, the binding interaction, such as a non-covalent interaction, between the binding partner C and at least one binding site Z is reversible.

In some embodiments, the reversible bond may be mediated by the presence of a substance, such as a competitive reagent (also referred to as an eluting reagent), that is or contains a binding site that is also capable of binding to at least one Z binding site. Typically, a substance (eg, a competitive reagent) may act as a competitor. For example, in some embodiments, the binding partner C dissociates from at least one Z binding site in accordance with its dissociation rate. In some aspects, after dissociation, the binding partner C can (i) re-bind to at least one Z binding site, or in some aspects (ii) re-binding to at least one Z binding site will be prevented if the substance, for example, is competitive the reagent first binds to one or more Z binding sites. In some aspects, the substance may have a higher binding affinity and/or be present at a high and/or sufficient concentration for the Z binding site present in the reagent and/or due to being present at higher concentrations than the binding partner C, thereby reducing the amount of attached and/or an associated bonding partner C from one or more bonding partners C. In some embodiments, the affinity of the substance (e.g., a competitive reagent) for at least one Z binding site is greater than the affinity of the binding partner C of the agent (e.g., a receptor binding agent or selective agent) for at least one Z binding site. In some embodiments, the association between the binding site Z of the reagent and the binding partner C of the agent (e.g., a receptor-binding agent or selective agent) can be reduced or reduced by the addition of a substance (e.g., a competitive reagent), thus making, in some aspects, the agent bond (eg, receptor-binding agent or selective agent) and the reagent is effectively reversible.

Reagents that can be used in such reversible systems are described and known in the art, see, for example, US Pat. No. 5,168,049; 5506121; 6103493; 7776562; 7981632; 8298782; 8735540; 9023604; and PCT International Published Application Nos. WO 2013/124474 and WO 2014/076277. Non-limiting examples of reagents and binding partners capable of forming a reversible interaction, as well as substances (eg, competitive reagents) capable of reversing such binding, are described below.

A. Reagent

In some embodiments, the reagent contains one or more Z binding sites that are capable of reversibly binding to C binding partners provided by the agent (eg, a receptor binding agent or a selective agent). In some embodiments, a reagent contains a plurality of Z binding sites, each of which is capable of specifically binding to a binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent), such that the reagent is capable of reversibly binding to a plurality of agents. (eg, a receptor-binding agent or a selective agent), for example, is a multimerization reagent and/or a particulate oligomeric reagent with one or more associated reagents. In some embodiments, the reagent is an oligomer or polymer of single molecules (eg, monomers) or complexes that make up a single molecule (eg, a tetramer), each containing at least one Z binding site. In some embodiments, the reagent contains at least two Z binding sites, at least three Z binding sites, at least four Z binding sites, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72 or more Z binding sites. All binding sites may be the same, or multiple binding sites may contain one or more different binding sites (eg, Z1, Z2, Z3, etc.). In some embodiments, the reagent is a particulate oligomeric reagent and contains at least 72, 120, 140, 200, 240, 280, 320, 360, 400, 440, 480, 520, 560, 600, 640, 680, 720 , 760, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or at least 100,000 Z binding sites.

In some embodiments, one or more agents (eg, receptor binding agents or selective agents) are linked, eg, reversibly linked to a reagent, eg, via one or more Z binding sites present on the reagent. In some cases, this results in agents (eg, receptor-binding agents or selective agents) located close to each other so that an avidity effect can occur if a target cell, having (at least two copies of) cell surface molecules, is contacted with an agent (eg, a receptor-binding agent or selective agent) that has one or more B binding sites capable of binding a particular molecule.

In some embodiments, two or more different agents (eg, receptor binding agents or selective agents) that are the same, i. contain the same binding site B, can be reversibly associated with the reagent. In some embodiments, two or more different agents containing the same B binding site are reversibly linked to a particulate oligomeric reagent. In some embodiments, at least two different (kinds of) agents (e.g., receptor binding agents or selective agents) may be used, and in some cases, three or four different (kinds of) agents, for example, two or more different receptor binding agents and/or a selective agent. For example, in some embodiments, the reagent may be reversibly linked to a first agent (eg, a receptor-binding agent or selective agent) containing a B1, B2, B3, or B4 region, etc. Bindings, and a second agent (eg, a receptor-binding agent or selective agent) containing a different binding site, for example, a different B1, B2, B3 or B4 binding site. In some cases, the binding site of the first agent and the second agent may be the same. For example, in some aspects, each of the at least two agents (eg, a receptor binding agent or a selective agent) may bind to the same molecule. In some cases, the binding site of the first agent and the second agent may be different. In some aspects, each of the at least two agents (eg, a receptor binding agent or a selective agent) may bind to a different molecule, such as a first molecule, a second molecule, and so on. In some cases, different molecules, such as cell surface molecules, may be present on the same target cell. In other cases, different molecules, such as cell surface molecules, may be present on different target cells that are present in the same cell population. In some instances, a third, fourth, and so forth agent (eg, receptor binding agent or selective agent) may be linked to the same reagent, each containing an additional different binding site.

In some embodiments, two or more different agents (eg, receptor binding agents or selective agents) contain the same binding partner C. In some embodiments, two or more different agents (eg, receptor binding agents or selective agents) contain different binding partners. In some aspects, the first agent (e.g., receptor binding agent or selective agent) may have a C1 binding partner that can specifically bind to the Z1 binding site present on the reagent, and the second agent (e.g., receptor binding agents or selective agent) may have a C2 binding partner that can specifically bind to the Z1 binding site or to the Z2 binding site present on the reagent. Thus, in some instances, the plurality of Z binding sites represented by a reagent include Z1 and Z2 binding sites that are capable of reversibly binding to C1 and C2 binding partners, respectively, represented by an agent (e.g., a receptor binding agent or a selective agent). In some embodiments, C1 and C2 are the same and/or Z1 and Z2 are the same. In other aspects, one or more of the plurality of Z binding sites may be different. In other cases, one or more of C's multiple binding partners may be different. The person skilled in the art can choose any combination of different C binding partners that are compatible with the reagent containing the Z binding sites, provided that each of the C binding partners is able to interact, for example, specifically bind to one of the Z binding sites.

In some embodiments, the reagent is a streptavidin, mutein or streptavidin analog, avidin, mutein or avidin analog (such as neutravidin), or a mixture thereof, wherein such reagent contains one or more Z binding sites for reversible bonding to a C binding partner. In some embodiments, binding partner C can be biotin, a biotin derivative or analog, or a streptavidin binding peptide or other molecule that is capable of specifically binding to streptavidin, a mutein or streptavidin analog, avidin or a mutein or avidin analog. In some embodiments, the reagent is or contains streptavidin, avidin, a streptavidin analog or mutein, or an avidin analog or mutein that reversibly binds biotin, a biotin analog, or a biologically active fragment thereof. In some embodiments, the reagent is or contains a streptavidin analog or mutein or an avidin analog or mutein that reversibly binds a streptavidin binding peptide. In some embodiments, the agent (eg, competitive reagent) can be biotin, a biotin derivative or analog, or a streptavidin-binding peptide capable of competing for binding with a C binding partner for one or more Z binding sites. In some embodiments, the binding partner C and the substance (eg, competitive reagent) are different, and the substance (eg, competitive reagent) exhibits a higher binding affinity for one or more Z binding sites compared to the affinity of the binding partner.

In some embodiments, the streptavidin may be wild-type streptavidin, muteins, or streptavidin analogs such as streptavidin-like polypeptides. Also in some aspects, avidin is wild-type avidin or muteins or avidin analogs such as neutravidin, deglycosylated arginine-modified avidin, which generally exhibits a more neutral pH and is available as an alternative to native avidin. Typically, deglycosylated, neutral forms of avidin include commercial forms such as "extravidin" sold by Sigma Aldrich or "Neutravidin" sold by, for example, Thermo Scientific or Invitrogen.

In some embodiments, the reagent is streptavidin or a mutein or a streptavidin analog. In some embodiments, the wild-type streptavidin (wt-streptavidin) has the amino acid sequence disclosed in Argarana et al, Nucleic Acids Res. 14 (1986) 1871-1882 (SEQ ID NO: 1). Typically, streptavidin exists in nature as a tetramer of four identical subunits, i.e. It is a homotetramer where each subunit contains one binding site for biotin, a biotin derivative or analog, or a biotin mimic. An exemplary streptavidin subunit sequence is the amino acid sequence shown in SEQ ID NO: 1, but such a sequence may also contain a sequence present in its homologues from another Streptomyces species. In particular, each streptavidin subunit can exhibit a strong binding affinity for biotin, with a dissociation constant (K _d ) on the order of about 10 ^-14 M. In some cases, streptavidin can exist as a monovalent tetramer in which only one of the four binding sites is functional (Howarth et al (2006) Nat Methods 3:267-73 Zhang et al (2015) Biochem Biophys Res Commun 463:1059-63)), a divalent tetramer in which two of the four binding sites are functional (Fairhead et al. (2013) J. Mol. biol. 426:199-214), or may be present in monomeric or dimeric form (Wu et al. (2005) J. biol. Chem. 280:23225- 31 Lim et al (2010) Biochemistry 50:8682-91).

In some embodiments, the streptavidin may be in any form, for example, wild-type or unmodified streptavidin, such as streptavidin from the species Streptomyces or a functional fragment thereof, which contains at least one functional subunit containing a binding site for biotin, a biotin derivative or analog or a biotin mimic, such as typically comprising at least one functional wild-type streptavidin subunit from Streptomyces avidinii as set forth in SEQ ID NO: 1, or a functional fragment thereof. For example, in some embodiments, the streptavidin may comprise a wild-type streptavidin fragment that is truncated at the N- and/or C-terminus. Such minimal streptavidins include any that start at the N-terminus in the region of amino acid positions 10-16 of SEQ ID NO: 1 and end at the C-terminus in the region of amino acid positions 133-142 of SEQ ID NO: 1. In some embodiments, a functionally active fragment streptavidin contains the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the implementation of streptavidin, such as shown in SEQ ID NO: 2, may additionally contain an N-terminal methionine at the position corresponding to Ala13 with the numbering given in SEQ ID NO: 1. Reference to the position of residues in streptavidin or streptavidin muteins is given with reference to the numbering of the residues in SEQ ID NO: 1.

In some aspects, streptavidin muteins comprise polypeptides that differ from the sequence of unmodified streptavidin or wild-type streptavidin by one or more amino acid substitutions, deletions, or additions, but which contain at least one functional subunit containing a binding site for biotin, a biotin derivative or analog, or streptavidin -binding peptide. In some aspects, streptavidin-like polypeptides and streptavidin muteins can be polypeptides that are substantially immunologically equivalent to wild-type streptavidin, and in particular are capable of binding biotin, biotin derivatives, or biotin analogs with the same or different affinity than wt-streptavidin. In some cases, streptavidin-like polypeptides or streptavidin muteins may contain amino acids that are not part of the wild-type streptavidin, or they may contain only a portion of the wild-type streptavidin. In some embodiments, the streptavidin-like polypeptides are polypeptides that are not identical to wild-type streptavidin because the host does not have the enzymes required to transform the host-produced polypeptide into the wild-type streptavidin structure. In some embodiments, streptavidin may also be present as streptavidin tetramers and streptavidin dimers, in particular streptavidin homotetramers, streptavidin homodimers, streptavidin heterotetramers, and streptavidin heterodimers. Typically, each subunit typically has a binding site for biotin or biotin analogs, or for streptavidin-binding peptides. Examples of streptavidins or streptavidin muteins are given, for example, in WO 86/02077, DE 19641876 A1, US 6022951, WO 98/40396 or WO 96/24606.

In some embodiments, the implementation of biotin is usually used in its natural d-sterioisomeric form, i. D-biotin. In some embodiments, the biotin is D-biotin. In specific embodiments, examples of biotin analogs and/or derivatives include, but are not limited to, D-biotin, N-ketone biotin analog, biotin ketone analog, biotin N-azide analog, biotin azide analog, biotin N-acylazide analog, NBD-GABA biotin analog, 1,2-diamine biotin analog, N-alkyne biotin analog, tetrathiol biotin analog, N-hydroxysuccinimide-iminobiotin, iminobiotin, amidobiotin N-hydroxysuccinimide-iminobiotin, amidobiotins, destiobiotin, biotinsulfone, caproylamidobiotin and biocytin. In some aspects, the biotin analogs are or contain biotinsulfone, 2'-thiobiotin, 2'-iminobiotin, d-desthiobiotin, dl-desthiobiotin, dl-desthiobiotin methyl ester, and other imidazolidone derivatives and derivatives described in Green, N.M. (1975) in Advances in Protein Chemistr (Anson, M.L. and Edsell, J.T. Eds), Vol. 29, pp. 85-133, Academic Press, New York. In some embodiments, the biotin analog or derivative is D-biotin, destiobiotin, and/or iminobiotin.

In some embodiments, the streptavidin mutein may contain amino acids that are not part of the unmodified streptavidin or wild-type streptavidin, or may contain only a portion of the wild-type streptavidin or unmodified streptavidin. In some embodiments, the streptavidin mutein comprises at least one subunit that may have one more amino acid substitution(s) than an unmodified or wild-type streptavidin subunit, for example, compared to the wild-type streptavidin subunit shown in SEQ ID NO: 1, or a functionally active fragment thereof, such as those shown in SEQ ID NO: 2. In some embodiments, at least one streptavidin mutein subunit may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid differences compared to wild-type streptavidin or unmodified streptavidin and/or contains at least one subunit containing an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical sequences with amino sequence an acid of SEQ ID NO: 1, 2, or 59, wherein such streptavidin mutein exhibits functional activity to bind biotin, a biotin derivative or analog, or a biotin mimetic. In some embodiments, amino acid substitutions (substitutions) are conservative or non-conservative mutations. Examples of streptavidin muteins are known in the art, see, for example, US Pat. No. 5,168,049; 5506121; 6022951; 6156493; 6165750; 6103493; or 6368813; or PCT International Published Application No. WO 2014/076277.

In some embodiments, the streptavidin or streptavidin mutein comprises proteins containing one or more than one functional subunit containing one or more Z binding sites for biotin, a biotin derivative or analog, or a streptavidin-binding peptide, e.g., two or more, three or more, four or more, and in some cases 5, 6, 7, 8, 9, 10, 11, 12 or more functional subunits. In some embodiments, the streptavidin or streptavidin mutein may contain a monomer; dimer, including heterodimer or homodimer; tetramer, including homotetramer, heterotetramer, monovalent tetramer or divalent tetramer; or may contain higher order multimers or oligomers thereof.

In some embodiments, the binding affinity, e.g., the dissociation constant (K _d ) of streptavidin or streptavidin mutein for the peptide ligand binding partner is less than 1×10 ^-4 M, 5×10 ^-4 M, 1×10 ^-5 M, 5 ×10 ^-5 M, 1 × 10 ^-6 M, 5 × 10 ^-6 M or 1 × 10 ^-7 M, but usually more than 1 × 10 ^-13 M, 1 × 10 ^-12 M or 1 × 10 ^-11 M. For example, peptide sequences (Strep-tags) such as those disclosed in US Pat. No. 5,506,121 can act as mimics of biotin and exhibit binding affinity for streptavidin, for example, with a K _d between approximately 10 ^-4 and 10 ^-5 M. In some cases, binding affinity can be further improved by generating a mutation in the streptavidin molecule, see, for example, US Pat. No. 6,103,493 or PCT International Published Application No. WO 2014/076277. In some embodiments, binding affinity can be determined using methods known in the art, such as any described herein.

In some embodiments, the reagent, such as streptavidin or a streptavidin mutein, exhibits binding affinity for the binding partner of the ligand of the peptide, wherein the binding partner of the ligand of the peptide can be a binding partner C present in the agent (e.g., a receptor binding agent or selective agent) . In some embodiments, the peptide sequence contains a sequence with the general formula given in SEQ ID NO: 9, for example, contains the sequence given in SEQ ID NO: 10. In some embodiments, the peptide sequence has the general formula given in SEQ ID NO: 11 , such as shown in SEQ ID NO: 12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag®, shown in SEQ ID NO: 7). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also referred to as Strep-tag® II, shown in SEQ ID NO: 8). In some embodiments, the implementation of the peptide ligand contains a sequential arrangement of at least two streptavidin-binding modules, while the distance between the two modules is at least 0 and not more than 50 amino acids, while one binding module has 3-8 amino acids and contains at least least the sequence of His-Pro-Xaa (SEQ ID NO: 9), where Xaa is glutamine, asparagine or methionine, and while the other binding module has the same or a different streptavidin peptide ligand, such as shown in SEQ ID NO: 11 (See, for example, PCT International Published Application No. WO 02/077018; US Patent No. 7981632). In some embodiments, the peptide ligand comprises a sequence having the formula given in any of SEQ ID NOs: 13 or 14. In some embodiments, the peptide ligand has an amino acid sequence given in any of SEQ ID NOs: 15-19. In most cases, all of these streptavidin-binding peptides bind to the same binding site, namely the biotin binding site of streptavidin. when one or more of these streptavidin-binding peptides are used as C binding partners, e.g., C1 and C2, the multimerization reagent and/or particulate oligomeric reagents associated with one or more agents via binding partner C typically consist of one or more streptavidin muteins.

In some embodiments, the reagent is or contains a streptavidin mutein. In some embodiments, the streptavidin muteins contain one or more mutations (eg, amino acid substitutions) compared to the wild-type streptavidin set forth in SEQ ID NO: 1, or a biologically active portion thereof. For example, the biologically active portions of streptavidin may contain streptavidin variants shorter at the N- and/or C-terminus, which in some cases are referred to as minimal streptavidin. In some embodiments, a minimal N-terminal truncated streptavidin at which any of the mutations can be made begins at the N-terminus in the region of amino acid positions 10-16 and ends at the C-terminus in the region of amino acid positions 133-142 compared to the sequence, shown in SEQ ID NO: 1. In some embodiments, a streptavidin with a truncated N-terminus at which any of the mutations can be made contains the amino acid sequence shown in SEQ ID NO: 2 or 59. In some embodiments, the minimum streptavidin contains the amino acid sequence from position Ala13 to Ser139 and optionally has an N-terminal methionine residue instead of Ala13. For the purposes of this document, amino acid position numbering throughout refers to the wt-streptavidin numbering given in SEQ ID NO: 1 (e.g., Argarana et al. Nucleic Acids Res. 14 (1986), 1871-1882, see also FIG. 9).

In some embodiments, the streptavidin mutein is a mutant as described in US Pat. SEQ ID NO: 1. In some embodiments, the streptavidin mutein comprises a mutation at one or more of residues 44, 45, 46, and/or 47. In some embodiments, the streptavidin mutein comprises a replacement of Glu at position 44 of the wild-type streptavidin with a hydrophobic aliphatic amino acid, for example, Val, Ala, Ile, or Leu, any amino acid at position 45, an aliphatic amino acid such as a hydrophobic aliphatic amino acid at position 46, and/or a substitution of Val at position 47 with a basic amino acid, e.g. Arg or Lys, e.g. usually Arg. In some embodiments, position 46 is Ala and/or position 47 is Arg and/or position 44 is Val or Ile. In some embodiments, the streptavidin mutant comprises Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ residues such as those shown in exemplary streptavidin muteins containing the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or 60 (also known as a streptavidin 1 mutant, SAM1). In some embodiments, the streptavidin mutein contains Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ residues, such as those shown in exemplary streptavidin muteins containing the amino acid sequence shown in SEQ ID NO: 5, 6, or 61 (also known as SAM2). In some instances, such streptavidin muteins are described, for example, in US Pat. No. 6,103,493 and sold under the tradename Strep-Tactin®.

In some embodiments, the streptavidin mutein is a mutant as described in PCT International Published Application No. WO 2014/076277. In some embodiments, the streptavidin mutein contains at least two cysteine residues in the region of amino acid positions 44-53, with reference to the amino acid positions shown in SEQ ID NO: 1. In some embodiments, cysteine residues are present at positions 45 and 52, creating a disulfide bridge linking these amino acids. In such an embodiment, amino acid 44 is typically glycine or alanine, amino acid 46 is typically alanine or glycine, and amino acid 47 is typically arginine. In some embodiments, the streptavidin mutein contains at least one mutation or amino acid difference in the region of amino acid residues 115-121 with reference to the amino acid positions shown in SEQ ID NO: 1. In some embodiments, the streptavidin mutein contains at least one mutation in the amino acid positions 117, 120 and 121 and/or deletion of amino acids 118 and 119 and substitution of at least amino acid position 121.

In some embodiments, the streptavidin mutein contains a mutation at position corresponding to position 117, where the mutation may be a large hydrophobic residue, such as Trp, Tyr, or Phe, or a charged residue, such as Glu, Asp, or Arg, or a hydrophilic residue, such as Asn or Gln, or in some cases hydrophobic Leu, Met or Ala residues, or polar Thr, Ser or His residues. In some embodiments, a mutation at position 117 is combined with a mutation at position corresponding to position 120, which mutation may be a small residue, such as Ser or Ala or Gly, and a mutation at position corresponding to position 121, which mutation may be a hydrophobic moiety, eg a bulky hydrophobic moiety, eg Trp, Tyr or Phe. In some embodiments, a mutation at position 117 is combined with a mutation at position 120 of the wild-type streptavidin shown in SEQ ID NO:1 or a biologically active fragment thereof, which mutation may be a hydrophobic residue such as Leu, Ile, Met, or Val or usually Tyr or Phe, and a mutation at the position corresponding to position 121 compared to the positions of the wild-type streptavidin shown in SEQ ID NO: 1 or its biologically active fragment, and this mutation may be a small residue, for example, Gly, Ala or Ser, or with Gln or a hydrophobic residue such as Leu, Val, Ile, Trp, Tyr, Phe or Met. In some embodiments, such muteins may also contain Val44-Thr45-Ala46-Arg47 residues or Ile44-Gly45-Ala46-Arg47 residues. In some embodiments, the streptavidin mutein contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120, and Tyr121. In some embodiments, the streptavidin mutein contains the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 27 or SEQ ID NO: 28, contains residues Val44, Thr45, Ala46, Arg47, Glu117, Gly120 and Tyr121 and exhibits functional binding activity to biotin, biotin analog or streptavidin-binding peptide.

In some embodiments, the molecule, eg, streptavidin or streptavidin mutein, has approximately 5-30 primary amines, which in some cases may include an N-terminal amine and/or one or more lysine residues. In specific embodiments, the molecule is a tetramer of streptavidin or a streptavidin mutein, including any of the described streptavidin muteins, which as a tetramer contains more than 5 primary amines, e.g., typically 5-40 or 15-35, e.g., typically about 15, 16, 17, 18, 19, 20, 21, 22, 23, 14, 25, 26, 27, 28, 29, 30, 31, 32 or more primary amines. In some embodiments, the molecule is streptavidin or a streptavidin mutein, or a truncated fragment thereof, such as any of the molecules described. In specific embodiments, the molecule is a tetramer of streptavidin or a streptavidin mutein containing the sequence shown in any of SEQ ID NOs: 2, 4, 6, 27, 59, 60, or 61, which as a tetramer is a molecule that contains 20 primary amines, including 1 N-terminal amine and 4 lysines per monomer.

In some embodiments, the streptavidin mutein may contain any of the above mutations in any combination, and the resulting streptavidin mutein may exhibit a binding affinity characterized by a dissociation constant (K _d ) that is 3.7×10 ^-5 M or less for the peptide ligand ( Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also called Strep-tag®, listed in SEQ ID NO: 7) and/or which is 7.1 x 10 ^-5 M or less for the peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also called Strep-tag® II, listed in SEQ ID NO: 8) and/or which is 7.0×10 ^-5 M, 6.0 ×10 ^-5 M, 5.0 × 10 ^-5 M, 4.0 × 10 ^-5 M, 3.0 × 10 ^-5 M, 2.0 × 10 ^-5 M, 1.0 × 10 ^-5 M , 9.0×10 ^-6 M, 8.0×10 ^-6 M, 7.0×10 ^-6 M, 6.0×10 ^-6 M, 5.0×10 ^-6 M, 4.0× 10 ^-6 M, 3.0×10 ^-6 M, 2.0×10 ^-6 M, 1.0×10 ^-6 M, 9.0×10 ^-7 M, 8.0×10 ^-7 M, 7.0×10 ^-7 M, 6.0×10 ^-7 M, 5.0×10 ^-7 M, 4.0×10 ^-7 M, 3.0×10 ^-7 M, 2.0×10 ^-7 M or 1.0×10 ^-7 M or less, but usually greater than 1×10 ^-13 M, 1×10 ^-12 M or 1×10 ^-11 M for any of peptide ligands as set forth in any one of SEQ ID NOs: 7-19.

In some embodiments, the streptavidin mutein may contain any of the above mutations in any combination, and the resulting streptavidin mutein may exhibit a binding affinity characterized by an association constant (K _a ) that is 2.7×10 ⁴ M ^-1 or more for the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also called Strep-tag®, listed in SEQ ID NO: 7) and/or which is 1.4×10 ⁴ M ^-1 or more for the ligand peptide (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also called Strep-tag® II, shown in SEQ ID NO: 8) and/or which is 1.43×10 ⁴ M ^-1 , 1 .67×10 ⁴ M ^-1 , 2×10 ⁴ M ^-1 , 3.33×10 ⁴ M ^-1 , 5×10 ⁴ M ^-1 , 1×10 ⁵ M ^-1 , 1.11×10 ⁵ M ^-1 , 1.25×10 ⁵ M ^-1 , 1.43×10 ⁵ M ^-1 , 1.67×10 ⁵ M ^-1 , 2×10 ⁵ M ^-1 , 3.33×10 ⁵ M ^-1 , 5×10 ⁵ M ^-1 , 1×10 ⁶ M ^-1 , 1.11×10 ⁶ M ^-1 , 1.25×10 ⁶ M ^-1 , 1.43×10 ⁶ M ^-1 , 1.67 ×10 ⁶ M ^-1 , 2 × 10 ⁶ M ^-1 , 3.33 × 10 ⁶ M ^-1 , 5 × 10 ⁶ M ^-1 , 1 × 10 ⁷ M ^-1 or more, but usually less than 1 × 10 ¹³ M ^-1 , 1×10 ¹² M ^-1 or 1×10 ¹¹ M ^-1 for any of the peptide ligands listed in any of SEQ ID NOs:7-19.

In some embodiments, the streptavidin mutein exhibits an amino acid sequence shown in any of SEQ ID NOs: 3-6, 27, 28, 60, or 61, or an amino acid sequence that exhibits at least 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence shown in any of SEQ ID NOs: 3-6, 27, 28, 60, or 61 and demonstrates binding affinity , characterized by a dissociation constant (K _d ) that is 3.7×10 ^-5 M or less for the peptide ligand (Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; also called Strep-tag®, is given in SEQ ID NO: 7) and/or which is 7.1 x 10 ^-5 M or less for a peptide ligand (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys; also called Strep-tag® II, shown in SEQ ID NO: 8) and/or which is 7.0×10 ^-5 M, 6.0×10 ^-5 M, 5.0×10 ^-5 M, 4.0×10 ^-5 M, 3, 0×10 ^-5 M, 2.0×10 ^-5 M, 1.0×10 ^-5 M, 9.0×10 ^-6 M, 8.0×10 ^-6 M, 7.0×10 ^-6 M, 6.0×10 ^-6 M, 5.0×10 ^-6 M, 4.0 ×10 ^-6 M, 3.0 × 10 ^-6 M, 2.0 × 10 ^-6 M, 1.0 × 10 ^-6 M, 9.0 × 10 ^-7 M, 8.0 × 10 ^-7 M , 7.0×10 ^-7 M, 6.0×10 ^-7 M, 5.0×10 ^-7 M, 4.0×10 ^-7 M, 3.0×10 ^-7 M, 2.0× 10 ^-7 M or 1.0 x 10 ^-7 M or less, but usually greater than 1 x 10 ^-13 M, 1 x 10 ^-12 M or 1 x 10 ^-11 M for any of the peptide ligands listed in any of SEQ ID NOS: 7-19.

In some embodiments, the streptavidin mutein also exhibits binding to other streptavidin ligands such as, but not limited to, biotin, iminobiotin, lipoic acid, dethiobiotin, diaminobiotin, HABA and/or dimethyl-HABA. In some embodiments, the streptavidin mutein exhibits a binding affinity for another streptavidin ligand, such as biotin or destiobiotin, greater than the binding affinity of the streptavidin mutein for a biotin mimetic peptide ligand, such as set forth in any of SEQ ID NOs: 7-19. In some embodiments, the streptavidin mutein exhibits a binding affinity for another streptavidin ligand, such as biotin or destiobiotin, the same, about the same as, or lower than the binding affinity of the streptavidin mutein for a biotin mimic peptide ligand, such as set forth in any of SEQ ID NO: 7 -19. In some embodiments, biotin or a biotin analog or derivative (eg, dethiobiotin) can be used as a competitive reagent in the present methods. For example, the interaction of a streptavidin mutein designated Strept-tactin® (for example, containing the sequence given in SEQ ID NO: 4 or 60) with a peptide ligand designated STREP-TAG® II (for example, given in SEQ ID NO: 8) is characterized by affinity binding to K_d approximately 10^-6 M compared to approximately 10^-13 M for biotin-streptavidin interaction. In some cases, biotin, which can bind with high affinity to Strep-Tactin® with K_dbetween or between approximately 10^-ten and 10^-13 M, can compete with STREP-TAG ® II for the binding site.

In some instances, the reagent contains at least two K chelating groups, which may be capable of bonding to the transition metal ion. In some embodiments, the reagent may be capable of binding to an oligohistidine affinity tag, glutathione-S-transferase, calmodulin or analogue thereof, calmodulin binding peptide (CBP), FLAG peptide, HA-tag, maltose-binding protein (MBP), HSV epitope, a myc epitope and/or a biotinylated carrier protein.

In some embodiments, the reagent is an oligomer or polymer. In some embodiments, an oligomer or polymer can be created by directly or indirectly linking individual molecules of a protein as it exists in nature, or by directly or indirectly linking individual molecules of a monomer or complex of subunits that make up a single molecule (e.g., direct or indirect linking of dimers, trimers, tetramers, etc. of the protein as it exists in nature). For example, in some embodiments, a tetrameric streptavidin or avidin may be referred to as a single molecule or the smallest building block of the respective oligomer or polymer. In specific embodiments, the tetrameric homodimer or heterodimer of streptavidin or avidin may be referred to as a single molecule or the smallest building block of the respective oligomer or polymer. In some embodiments, the oligomer or polymer may have a bond of at least 2 individual protein molecules (e.g., is a 2-mer), or may be at least 3-mer, 4-mer, 5-mer, 6-mer, 7 -mer, 8-mer, 9-mer, 10-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer , 20-mer, 25-mer, 30-mer, 35-mer, 40-mer, 45-mer or 50-mer of individual protein molecules (eg monomers, tetramers). In some embodiments, the oligomer may be at least 100-mer, 200-mer, 300-mer, 400-mer, 500-mer, 1000-mer, 1500-mer, 2000-mer, 2500-mer, 3000-mer, or at least 3500-mers from individual protein molecules. In some embodiments, the reagent is a particulate oligomeric reagent as described in section II(A)(1) or (2), or is a particulate oligomeric reagent that is prepared using the methods described in section II(B). )(3).

Oligomers can be created using any of the methods known in the art, for example, any of those described in US Published Application No. US 2004/0082012. In some embodiments, the implementation of the oligomer or polymer contains two or more separate molecules that can be sewn, for example, using a polysaccharide or bifunctional linker.

In some embodiments, the implementation of the oligomer or polymer is obtained by cross-linking individual molecules or a complex of subunits that make up a single molecule, in the presence of a polysaccharide. In some embodiments, the implementation of oligomers or polymers can be obtained by introducing carboxyl residues in a polysaccharide, such as dextran. In some aspects, individual reactant molecules (eg, monomers, tetramers) can be coupled via the primary amino groups of the internal lysine residues and/or the free N-terminus to the carboxyl groups in the dextran backbone using conventional carbodiimide chemistry. In some embodiments, the compound reaction is carried out at a molar ratio of about 60 moles of individual reactant molecules (eg, monomers, tetramers) per mole of dextran.

In some embodiments, the reagent is an oligomer or polymer of one or more of streptavidin or avidin, or any of a streptavidin analog or mutein or an avidin analog or mutein (eg, neutravidin). In some embodiments, the Z binding site is a natural biotin binding site of avidin or streptavidin, for which there can be up to four binding sites in a single molecule (for example, a tetramer contains four Z binding sites), due to which the homotetramer can contain up to 4 identical binding sites , i.e. Z1, while the hetero-tetramer may contain up to 4 binding sites, which may be different, for example, contains Z1 and Z2. In some embodiments, the oligomer is created or derived from multiple individual molecules (e.g., multiple homotetramers) of the same streptavidin, streptavidin mutein, avidin, or avidin mutein, in which case each Z binding site, e.g., Z1, of the oligomer is the same. For example, in some cases, the oligomer may contain multiple Z1 binding sites, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more Z1 binding sites. In some embodiments, the oligomer is made from or derived from a plurality of individual molecules, which may be hetero-tetramers of streptavidin, a streptavidin mutein, avidin, or an avidin mutein, and/or from a plurality of two or more different individual molecules (e.g., different homotetramers) of streptavidin, a streptavidin mutein , avidin, or avidin mutein that differ in their Z binding sites, eg Z1 and Z2, in which case many different Z binding sites may be present in the oligomer, eg Z1 and Z2. For example, in some cases, the oligomer may contain a plurality of Z1 binding sites and a plurality of Z binding sites, which in combination may contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50 or more merged Z1 and Z2 binding sites.

In some cases, the corresponding oligomer or polymer can be crosslinked with a polysaccharide. In one embodiment, oligomers or polymers of streptavidin or avidin or streptavidin or avidin analogues (e.g., neutravidin) can be prepared in the first step by introducing carboxyl residues into a polysaccharide, e.g., dextran, essentially as described in Noguchi, A, et al, Bioconjugate Chemistry (1992) 3,132-137. In some such aspects, in a second step, streptavidin or avidin or their analogs can then be coupled via the primary amino groups of the internal lysine residue and/or the free N-terminus to carboxyl groups in the dextran backbone using conventional carbodiimide chemistry. In some instances, cross-linked oligomers or polymers of streptavidin or avidin or any of the streptavidin or avidin analogs can also be prepared by cross-linking through bifunctional linker molecules such as glutardialdehyde or other methods described in the art.

In some embodiments, the implementation of the oligomer or polymer is obtained by cross-linking individual molecules or a complex of subunits that make up a single molecule, using a bifunctional linker or other chemical linker such as glutardialdehyde, or using other methods known in this field. In some aspects, cross-linked oligomers or polymers of streptavidin or avidin or any of the mutein or analog of streptavidin or avidin can be obtained by cross-linking individual streptavidin or avidin molecules through bifunctional molecules serving as a linker, such as glutardialdehyde, or using other methods described in this areas. For example, it is possible to create oligomers of streptavidin muteins by introducing thiol groups into the streptavidin mutein (this can be done, for example, by reacting the streptavidin mutein with 2-iminothiolane (Trot's reagent) and by activating, for example, in a separate reaction, the amino groups available in the streptavidin mutein In some embodiments, this amino group activation can be achieved by reacting a streptavidin mutein with a commercially available heterobifunctional crosslinker such as sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo SMCC) or succinimidyl-6-[(β-maleimidopropionamido )hexanoate (SMPH) In some such embodiments, the two reaction products thus obtained are stirred together, typically resulting in the reaction of the thiol groups contained in one batch of modified streptavidin mutein with activated (e.g., via maleimide functionality) amino acids of another lots of modified m streptavidin utein. In some cases, this reaction produces streptavidin mutein multimers/oligomers. These oligomers may have any suitable number of individual molecules, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 100, 200, 300, 400, 500, 1000 , 1500, 2000, 2500, 3000, 3500, 4,000 or more, and the degree of oligomerization can be changed according to the reaction conditions.

In some embodiments, an oligomeric or polymeric reagent can be isolated by size exclusion chromatography, and any desired fraction can be used as a reagent. For example, in some embodiments, after reacting the modified streptavidin mutein in the presence of 2-iminothiolane and a heterobifunctional crosslinker such as sulfo SMCC, the oligomeric or polymeric reagent can be isolated by size exclusion chromatography and any desired fraction can be used as the reagent. In some embodiments, the implementation of the oligomers do not (and should not have) a single molecular weight, but they can be observed statistical distribution of mass, such as a Gaussian distribution. In some cases, any oligomer with more than three streptavidin or mutein tetramers, e.g. homotetramers or heterotetramers, e.g. typically 3-50 tetramers, e.g. homotetramers or heterotetramers, 10-40 tetramers, e.g. homotetramers or heterotetramers, or 25-35 tetramers, eg homotetramers or heterotetramers. The oligomers may have, for example, 3-25 streptavidin mutein tetramers, eg homotetramers or heterotetramers. In some aspects, when the streptavidin muteins have a molecular weight of about 50 kDa, the oligomers can have a molecular weight of from about 150 kDa to about 2000 kDa, from about 150 kDa to about 1500 kDa, from about 150 kDa to about 1250 kDa, from about 150 kDa to about 1000 kDa , from about 150 kDa to about 500 kDa, or from about 150 kDa to about 300 kDa, from about 300 kDa to about 2000 kDa, from about 300 kDa to about 1500 kDa, from about 300 kDa to about 1250 kDa, from about 300 kDa up to 1000 kDa, from about 300 kDa to about 500 kDa, from about 500 kDa to about 2000 kDa, from about 500 kDa to about 1500 kDa, from about 500 kDa to about 1250 kDa, from about 500 kDa to about 1000 kDa, from about 1000 kDa to about 2000 kDa, from about 1000 kDa to about 1500 kDa, from about 1000 kDa to about 1250 kDa, from about 1250 kDa to about 2000 kDa, or from about 1500 kDa to about 2000 kDa. In some embodiments, the implementation of the oligomers have a molecular weight of more than 2000 kDa. Typically, due to the fact that each streptavidin/mutein molecule has four biotin binding sites, such a reagent can provide 12-160 Z binding sites, eg 12-160 or more Z binding sites. In some embodiments, the implementation of the oligomers are soluble reagents.

1. Oligomeric Particulate Reagents

Provided herein are particulate oligomeric reagents that consist of and/or contain a plurality of molecules, such as streptavidin tetramers or streptavidin mutein. In some embodiments, the particulate oligomeric reactants are soluble reactants. In some embodiments, the particulate oligomeric reagents provided herein contain at least one binding site that reversibly binds or is capable of reversibly binding to one or more agents, for example, a stimulating agent and/or a selective agent. In some embodiments, the particulate oligomeric reagents provided herein comprise a plurality of binding sites that are capable of reversibly binding to one or more agents, such as a site on a binding partner, such as binding partner C, that is linked to one or more agents. In some embodiments, the particulate oligomeric reagents are reversibly linked to one or more agents. In particular embodiments, the particulate oligomeric reagent is an oligomeric particle that is produced, prepared, or created by any of the methods described in section II(B).

In some embodiments, the particulate oligomeric reagents provided herein are composed of and/or contain oligomerized molecules that are proteins, polypeptides, peptides, and/or molecules that contain or include one or more amino acids. In some embodiments, the particulate oligomeric reagents provided herein comprise and/or consist of oligomerized molecules that contain a plurality of binding sites that are capable of binding to one or more agents, such as a receptor binding agent. In some embodiments, the particulate oligomeric reagent provided herein contains a plurality of binding sites that are capable of binding to an agent as described in section II(B)(4) and/or section II(B)(5). In some embodiments, the particulate oligomeric reagents provided herein comprise a plurality of binding sites that bind or are capable of binding to one or more agents at a site within a binding partner, e.g., a binding partner C, that is linked to one or more agents. In specific embodiments, a molecule that is oligomerized contains a plurality of binding sites that are capable of binding to a binding partner C, which is described in section II(A). In some embodiments, the molecule that is oligomerized is or contains streptavidin, a mutein, or a streptavidin analog, avidin, a mutein, or an avidin analog (eg, neutravidin). In some embodiments, streptavidin is a tetramer in its native state. Thus, in some embodiments, the molecule is a tetramer of streptavidin, mutein, or a streptavidin analog, avidin, mutein, or avidin analog (eg, neutravidin). In particular embodiments, the particulate oligomeric reagent comprises a plurality of one or more of any of the reagents described in section II(A).

In particular embodiments, the particulate oligomeric reactants are sized by any suitable means known in the art. In some embodiments, the weight and/or molecular weight of the particulate oligomeric reagents is determined by any suitable means in the art, including, but not limited to, electrophoresis, such as SDS-PAGE, chromatography, such as size exclusion chromatography, or SEC, or mass spectrometry. In some embodiments, the size, eg, radius, of the particulate oligomeric reactant is determined using a dynamic light scattering (DLS) technique. In some embodiments, the size, such as the radius, is determined using a flow field fractionation technique (F4). In some embodiments, F4 can be used to separate and measure particles based on size, regardless of particle density. In some embodiments, the implementation of the particle size is measured using asymmetric fractionation in the flow in the flow field (AF4). In some embodiments, the implementation of the particle size can be determined by measuring the diameter or radius of the particles. In some embodiments, the implementation of the particle size can be determined by measuring the hydrodynamic radius or radius of gyration of the particles. In some embodiments, the implementation of the radius is determined using the method of dynamic light scattering. In some embodiments, the radius, such as the hydrodynamic radius and/or the Stokes radius, can be determined using a chromatography method, such as size exclusion chromatography sec).

In particular embodiments, the particulate oligomeric reagent provided herein has a radius, e.g., an average radius, of at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 55 nm, at least 60 nm, at least 65 nm , at least 70 nm, at least 75 nm, at least 80 nm, at least 85 nm, at least 90 nm, at least 95 nm, at least 100 nm, at least 105 nm, at least 110 nm, at least 115 nm, at least 120 nm, at least 125 nm, at least 130 nm, at least 135 nm, at least or at least 140 nm. In some embodiments, the particulate oligomeric reagent has a radius between 5 nm and 150 nm, between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 140 nm, between 85 nm and 135 nm, between 80 nm and 120 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, inclusive. In some embodiments, the particulate oligomeric reagent provided herein has a radius of about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, approximately 94 nm, approximately 95 nm, approximately 96 nm, approximately 97 nm, approximately 98 nm, approximately 99 nm, approximately 100 nm, approximately 101 nm, approximately 102 nm, approximately 103 nm, approximately 104 nm, approximately 105 nm , approximately 106 nm, approximately 107 nm, approximately 108 nm, approximately 109 nm, approximately 110 nm, approximately 111 nm, approximately 112 nm, approximately 113 nm, approximately 114 nm, or approximately 115 nm. In some embodiments, the particles have a radius between 80 nm and 115 nm, inclusive.

In some embodiments, the radius is the hydrodynamic radius, the radius of gyration, the Stokes radius, the Stokes-Einstein radius, and/or the effective radius of hydrated particles in solution. In some embodiments, the radius is the hydrodynamic radius. In some embodiments, the implementation of the radius is the Stokes radius. In particular embodiments, the hydrodynamic radius is the Stokes radius. In some embodiments, the radius is the average, median, and/or median radius of the plurality of particles.

In some embodiments, the particulate oligomeric reagent provided herein has a molecular weight of at least 2×10 ⁶ g/mol, 3×10 ⁶ g/mol, 5×10 ⁶ g/mol, 1×10 ⁷ g /mol, at least 5×10 ⁷ g/mol, at least 1×10 ⁸ g/mol, at least 1.25×10 ⁸ g/mol, at least 1.5×10 ⁸ g/mol , at least 2×10 ⁸ g/mol or at least 5×10 ⁸ g/mol. In some embodiments, the particulate oligomeric reagent provided herein has a molecular weight between 1×10 ⁶ g/mol and 1×10 ¹⁰ g/mol, 2×10 ⁶ g/mol and 1×10 ¹⁰ g/mol , between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol, between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 7.5×10 ⁷ g/mol and 2.5× 10 ⁸ g/mol, between 2.5×10 ⁷ g/mol and 2.75×10 ⁸ g/mol, between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, between 7.5×10 ⁷ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g/mol and 2×10 ⁸ g/mol, inclusive. In specific embodiments, the particulate oligomeric reagent provided herein has a molecular weight of about 7.5×10 ⁷ g/mol, about 8.0×10 ⁷ g/mol, about 9.0×10 ⁷ g/mol , approximately 1.0×10 ⁸ g/mol, approximately 1.1×10 ⁸ g/mol, approximately 1.2×10 ⁸ g/mol, approximately 1.3×10 ⁸ g/mol, approximately 1.4× 10 ⁸ g/mol, approximately 1.5×10 ⁸ g/mol, approximately 1.6×10 ⁸ g/mol, approximately 1.7×10 ⁸ g/mol, approximately 1.8×10 ⁸ g/mol, about 1.9×10 ⁸ g/mol, about 2.0×10 ⁸ g/mol, about 2.1×10 ⁸ g/mol, about 2.2×10 ⁸ g/mol, about 2.3×10 ⁸ g /mol, approximately 2.4×10 ⁸ g/mol or approximately 2.5×10 ⁸ g/mol. In some embodiments, the particulate oligomeric reagent provided herein has a molecular weight between 5×10 ⁷ g/mol and 2×10 ⁸ g/mol, inclusive.

In some embodiments, the particulate oligomeric reagent provided herein consists of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the particulate oligomeric reagent provided herein consists of and/or contains at least 100, at least 200, at least 300, at least 400, at least 500, at least 600 , at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600 , at least 1700, at least 1800, at least 1900, at least 2200, at least 2300, at least 2400, at least 2500, at least 2600, at least 2700, at least 2800 , at least 2900, at least 3000, at least 4000, at least 5000, at least 10000 or at least 20000 streptavidin tetramers or streptavidin mutein. In particular embodiments, the particulate oligomeric reagents provided herein comprise and/or consist of between 100 and 50,000, between 500 and 10,000, between 1,000 and 20,000, between 500 and 5,000, between 300 and 7,500, between 1,500 and 7,500, between 500 and 3500, between 1000 and 5000, between 1500 and 2500, between 1500 and 2500, between 2000 and 3000, between 2500 and 3500, between 2000 and 4000, or between 2000 and 5000 streptavidin tetramers or streptavidin mutein. In some embodiments, the particulate oligomeric reagent provided herein consists of and/or contains between about 2000 and 3500 streptavidin tetramers or streptavidin mutein.

In some embodiments, provided herein is a particulate oligomeric reagent that consists of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the particulate oligomeric reagent provided herein contains a plurality of binding sites that reversibly bind or are capable of reversibly binding to one or more agents, for example, a stimulating agent and/or a selective agent. In some embodiments, the implementation of the oligomeric particle has a radius between 25 nm and 150 nm, inclusive; a molecular weight between 2×10 ⁶ g/mol and 1×10 ¹⁰ g/mol; and/or between 500 and 10,000 tetramers of streptavidin or streptavidin mutein.

In specific embodiments, provided herein is a particulate oligomeric reagent that consists of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the particulate oligomeric reagent provided herein contains a plurality of binding sites that reversibly bind or are capable of reversibly binding to one or more agents, for example, a stimulating agent and/or a selective agent. In some embodiments, the implementation of the oligomeric particles have a radius, for example, the average radius, between 70 nm and 125 nm, inclusive; a molecular weight between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol, inclusive; and/or between 1000 and 5000 tetramers of streptavidin or streptavidin mutein, inclusive. In some embodiments, the particulate oligomeric reagent is associated, eg, reversibly associated, with one or more agents, such as an agent that binds to a molecule, eg, a receptor, on the surface of a cell. In some embodiments, the implementation of one or more agents are agents described in this document, for example, in section II-C-3. In some embodiments, the agent is an anti-CD3 and/or anti-CD28 antibody, or an antigen-binding fragment thereof, e.g., an antibody or an antigen-binding fragment thereof, which contains a binding partner, e.g., a streptavidin-binding peptide, e.g., Strep-tag ®II. In specific embodiments, the one or more agents is an anti-CD3 and/or anti-CD28 Fab containing a binding partner, eg a streptavidin binding peptide, eg Strep-tag® II.

In some embodiments, provided herein is a particulate oligomeric reagent that consists of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the particulate oligomeric reagent provided herein contains a plurality of binding sites that reversibly bind or are capable of reversibly binding to one or more agents, for example, a stimulating agent and/or a selective agent. In some embodiments, the implementation of the oligomeric particles have a radius, for example, the average radius, between 80 nm and 120 nm, inclusive; molecular weight, for example, an average molecular weight between 7.5×10 ⁶ g/mol and 2×10 ⁸ g/mol, inclusive; and/or an amount, eg an average amount, between 500 and 10,000 tetramers of streptavidin or streptavidin mutein, inclusive. In some embodiments, the particulate oligomeric reagent is associated, eg, reversibly associated, with one or more agents, such as an agent that binds to a molecule, eg, a receptor, on the surface of a cell. In some embodiments, the implementation of one or more agents are agents described in this document, for example, in section II-C-3. In some embodiments, the agent is an anti-CD3 and/or anti-CD28 Fab, eg a Fab that contains a binding partner, eg a streptavidin binding peptide, eg Strep-tag® II. In specific embodiments, the one or more agents is an anti-CD3 and/or anti-CD28 Fab containing a binding partner, eg a streptavidin binding peptide, eg Strep-tag® II.

2. Compositions of oligomeric reagents in the form of particles

Provided herein are compositions containing particulate oligomeric reagents, eg, a plurality of particulate oligomeric reagents, that are composed of and/or contain a plurality of molecules, eg, streptavidin tetramers or streptavidin mutein. In some embodiments, the composition provided herein contains a plurality of any of the particulate oligomeric reagents described herein. In particular embodiments, the composition comprises a plurality of any of the particulate oligomeric reagents described in section II(A)(1). In some embodiments, the composition comprises a plurality of particulate oligomeric reactants that are made, prepared, and/or created by any of the methods described in section II(B).

In specific embodiments, the implementation of the composition contains oligomeric reagents in the form of particles with an average, median and/or median size. In some embodiments, the composition contains oligomeric reagents in the form of particles with a mean, median, or median radius of at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least at least 50 nm, at least 55 nm, at least 60 nm, at least 65 nm, at least 70 nm, at least 75 nm, at least 80 nm, at least 85 nm, at least 90 nm, at least 95 nm, at least 100 nm, at least 105 nm, at least 110 nm, at least 115 nm, at least 120 nm, at least 125 nm, at least 130 nm, at least 135 nm, at least or at least 140 nm. In some embodiments, the composition contains oligomeric reagents in the form of particles with an average, median or median radius between 5 nm and 150 nm, between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 140 nm, between 85 nm and 135 nm, between 80 nm and 120 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, inclusive.

In some embodiments, the composition contains oligomeric reagents in the form of particles with a mean, median, or median radius of 90 nm ± 25 nm, 90 nm ± 20 nm, 90 nm ± 15 nm, 90 nm ± 10 nm, 90 nm ± 5 nm, 95 nm ± 25 nm, 95 nm ± 20 nm, 95 nm ± 15 nm, 95 nm ± 10 nm, 95 nm ± 5 nm, 97 nm ± 20 nm, 97 nm ± 15 nm, 97 nm ± 10 nm, 97 nm ± 5 nm.

In some embodiments, the composition comprises particulate oligomeric reactants with a mean, median, or median radius of about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, approximately 93 nm approximately 94 nm approximately 95 nm approximately 96 nm approximately 97 nm approximately 98 nm approximately 99 nm approximately 100 nm approximately 101 nm approximately 102 nm approximately 103 nm approximately 104 nm approximately 105 nm, approximately 106 nm, approximately 107 nm, approximately 108 nm, approximately 109 nm, approximately 110 nm, approximately 111 nm, approximately 112 nm, approximately 113 nm, approximately 114 nm or approximately 115 nm. In some embodiments, the implementation of the composition contains oligomeric reagents in the form of particles with an average, median or median radius between 80 nm and 115 nm, inclusive.

In some embodiments, the particulate oligomeric reagents of the composition have an average, median, or median molecular weight of at least 2×10 ⁶ g/mol, 3×10 ⁶ g/mol, 5×10 ⁶ g/mol, 1×10 ⁷ g /mol, at least 5×10 ⁷ g/mol, at least 1×10 ⁸ g/mol, at least 1.25×10 ⁸ g/mol, at least 1.5×10 ⁸ g/mol , at least 2×10 ⁸ g/mol or at least 5×10 ⁸ g/mol. In some embodiments, the particulate oligomeric reagents of the composition have an average, median, or median molecular weight between 1×10 ⁶ g/mol and 1×10 ¹⁰ g/mol, 2×10 ⁶ g/mol and 1×10 ¹⁰ g/mol , between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol, between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 7.5×10 ⁷ g/mol and 2.5× 10 ⁸ g/mol, between 2.5×10 ⁷ g/mol and 2.75×10 ⁸ g/mol, between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, between 7.5×10 ⁷ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g/mol and 2×10 ⁸ g/mol, inclusive. In particular embodiments, the particulate oligomeric reagent compositions have an average, median, or median molecular weight of about 7.5×10 ⁷ g/mol, about 8.0×10 ⁷ g/mol, about 9.0×10 ⁷ g/mol , approximately 1.0×10 ⁸ g/mol, approximately 1.1×10 ⁸ g/mol, approximately 1.2×10 ⁸ g/mol, approximately 1.3×10 ⁸ g/mol, approximately 1.4× 10 ⁸ g/mol, approximately 1.5×10 ⁸ g/mol, approximately 1.6×10 ⁸ g/mol, approximately 1.7×10 ⁸ g/mol, approximately 1.8×10 ⁸ g/mol, about 1.9×10 ⁸ g/mol, about 2.0×10 ⁸ g/mol, about 2.1×10 ⁸ g/mol, about 2.2×10 ⁸ g/mol, about 2.3×10 ⁸ g /mol, approximately 2.4×10 ⁸ g/mol or approximately 2.5×10 ⁸ g/mol. In some embodiments, the implementation of the oligomeric reagents in the form of particles of the composition have an average, median or median molecular weight between 5×10 ⁷ g/mol and 2×10 ⁸ g/mol, inclusive.

In some embodiments, each of the oligomeric particulate reagents of the composition consists of and/or contains a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, the particulate oligomeric reactants of the composition are composed of and/or contain an average, median, or median amount of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600 at least 1700 at least 1800 at least 1900 at least 2200 at least 2300 at least 2400 at least 2500 at least 2600 at least 2700 at least 2800, at least 2900, at least 3000, at least 4000, at least 5000, at least 10000 or at least 20000 streptavidin tetramers or streptavidin mutein. In particular embodiments, the particulate oligomeric reactants compositions comprise and/or consist of an average, median, or median amount between 100 and 50,000, between 500 and 10,000, between 1,000 and 20,000, between 500 and 5,000, between 300 and 7,500, between 1,500 and 7500, between 500 and 3500, between 1000 and 5000, between 1500 and 2500, between 1500 and 2500, between 2000 and 3000, between 2500 and 3500, between 2000 and 4000, or between 2000 and 5000 streptavidin or mutein tetramers. In some embodiments, the particulate oligomeric reagents of the composition are composed of and/or contain an average, median, or median amount of between about 2000 and 3500 streptavidin tetramers or streptavidin mutein.

In some embodiments, the implementation of the composition contains oligomeric reagents in the form of particles with a distribution of sizes. In some embodiments, the particulate oligomeric reactants of the composition have a size distribution such that at least 70%, 80%, 90%, or 95% of the oligomeric particulate reactants of the composition have a size that is within ±100%, ±90% , ± 80%, ± 70%, ± 60%, ± 50%, ± 40%, ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, ± 0.1%, ± 0.01% or ± 0.001% of the median or average size of the oligomeric reagents in the form of particles of the composition. In some embodiments, size is measured in terms of radius, molecular weight, or number of molecules, such as streptavidin tetramers or streptavidin mutein, a particulate oligomeric reagent.

In some embodiments, at least 95% of the particulate oligomeric reagents of the composition have a radius that is within ±100%, ±90%, ±80%, ±70%, ±60%, ±50%, ±40%, ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, ±1%, ±0.5%, ±0.1%, ±0.01%, or ±0.001% of median or the average radius of the oligomeric reagents in the form of particles of the composition. In specific embodiments, at least 95% of the particulate oligomeric reagents of the composition have a radius that is between 10 nm and 250 nm, between 25 nm and 200 nm, between 50 and 150 nm, between 70 nm and 140 nm, between 70 and 130 nm, between 70 and 100 nm, between 80 and 110 nm, between 80 and 120 nm, between 80 and 115 nm, between 80 and 100 nm, between 90 and 120 nm, between 90 and 110 nm, between 100 nm and 120 nm, or between 85 and/or 115 nm, inclusive. In specific embodiments, at least 95% of the particulate oligomeric reactants of the composition have a radius that is within ±25%, ±20%, ±15%, ±10%, ±5%, or ±1% of the average radius of the oligomeric reactants in composition particles.

In specific embodiments, at least 95% of the particulate oligomeric reagents of the composition have a molecular weight that is within ±100%, ±90%, ±80%, ±70%, ±60%, ±50%, ±40% , ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, ± 0.1%, ± 0.01% or ± 0.001% median or average molecular weight of oligomeric reagents in the form of particles of the composition. In some embodiments, at least 95% of the particulate oligomeric reactants of the composition have a molecular weight between 2×10 ⁶ g/mol and 1×10 ¹⁰ g/mol, between 1×10 ⁶ g/mol and 1×10 ⁸ g/mol. mol, between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol, between 1×10 ⁸ g/mol and 1×10 ¹⁰ g/mol, between 1×10 ⁸ g/mol and 1×10 ⁹ g /mol, between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 1×10 ⁹ g/mol and 1×10 ¹⁰ g/mol, between 1×10 ⁷ g/mol and x 10 ⁸ g /mol, between 7.5×10 ⁷ g/mol and 2.5×10 ⁸ g/mol, between 5×10 ⁷ g/mol and 2.5×10 ⁸ g/mol, between 1×10 ⁸ g/ mol and 3×10 ⁸ g/mol, between 7.0×10 ⁷ g/mol and 3.0×10 ⁸ g/mol, or between 1×10 ⁸ g/mol and 2×10 ⁸ g/mol, inclusive. In some embodiments, at least 95% of the particulate oligomeric reagents of the composition have a molecular weight that is within ±25%, ±20%, ±15%, ±10%, ±5%, or ±1% of the average molecular weight of the oligomeric reagents in the form of particles of the composition.

In some embodiments, the oligomeric particles of the composition are comprised of a plurality of streptavidin or streptavidin mutein tetramers, and at least 95% of the oligomeric particulate reagents are comprised of tetramers within ±100%, ±90%, ±80%, ±70%, ± 60%, ± 50%, ± 40%, ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, ± 1%, ± 0.5%, ± 0.1% , ± 0.01% or ± 0.001% of the median or mean number of tetramers per particulate oligomeric reagent. In some embodiments, the oligomeric particles of the composition, at least 95% of the oligomeric particulate reactants, consist of between 100 and 50,000, between 500 and 10,000, between 1,000 and 20,000, between 1,000 and 5,000, between 5,000 and 10,000, between 10,000 and 15,000 , between 1500 and 4000, between 2000 and 4500, between 2500 and 5000, between 3000 and 5000, between 3500 and 5500, between 4000 and 6000, or between 1500 and 3500 streptavidin tetramers or streptavidin mutein. In some embodiments, at least 95% of the particulate oligomeric reactants of the composition consist of an amount of tetramers in the range of ±25%, ±20%, ±15%, ±10%, ±5%, or ±1% of the average molecular weight of the oligomeric reactants in composition particles.

In some embodiments, the particulate oligomeric reagent composition is stored for a period of time, such as after the particulate oligomeric reagents are made, prepared, and/or formulated, and before the addition of the agent, such as a receptor binding agent. In some embodiments, the particulate oligomeric reagent composition is stored in a neutral pH buffer. In some embodiments, the composition is stored as separate aliquots. In some embodiments, the particulate oligomeric reagent composition is stored at or below room temperature, at 4°C or below, at -20°C or below, or at -80°C or below. In some embodiments, the composition is stored for a period of time that is, approximately, or at least 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days , 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 27 weeks, 28 weeks, 30 weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44 weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 60 weeks, 70 weeks, 80 weeks, 90 weeks, 12 months, 16 months, 18 months, 24 months, 30 months, 36 months or more than 36 months. In specific embodiments, the particulate oligomeric reagent composition is stored at or below 4° C. for, for about, or for at least 1 week, 9 weeks, 27 weeks, or 46 weeks. In some embodiments, the particulate oligomeric reagent composition is stored at or below -80° C. for, for about, or for at least 1 week, 9 weeks, 27 weeks, or 46 weeks. In specific embodiments, the dimensions of the oligomeric particles during storage are stable, for example, the size does not change or increase by more than 25%, 20%, 15%, 10%, or 5%.

In particular embodiments, the particulate oligomeric reactants of the composition do not experience an increase in average, eg median, particle size during storage. In some embodiments, the composition is stored for a period of time and the particulate oligomeric reactants do not experience an average size increase of greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater than 25%, more than 30%, more than 40% or more than 50%. In specific embodiments, the composition is stored at or below about 4°C, at or below -20°C, or at or below -80°C for at least 9, 27, or 46 weeks and does not experience an average size increase greater than by 1%, 5% or 10%. In some embodiments, the implementation of the composition is stored at approximately -80°C.

In some embodiments, provided herein is a composition of particulate oligomeric reagents that consist of and/or contain a plurality of streptavidin or streptavidin mutein tetramers. In some embodiments, each of the oligomeric particulate reagents of the composition contains a plurality of binding sites that reversibly bind or are capable of reversibly binding to one or more agents, for example, a stimulating agent and/or a selective agent. In some embodiments, the particulate oligomeric reagents have a median, median, or median radius between 25 nm and 150 nm; average, median or median molecular weight between 2×10 ⁶ g/mol and 1×10 ¹⁰ g/mol; and/or an average, median or median amount between 500 and 10,000 streptavidin or streptavidin mutein tetramers. In some embodiments, at least 70%, 80%, 90%, or 95% of the oligomeric particulate reactants of the composition have a radius, molecular weight, or number of tetramers that are within ±100%, ±90%, ±80%, ± 70%, ± 60%, ± 50%, ± 40%, ± 30%, ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, ± 1% mean, median or median radius, molecular the mass or number of tetramers of oligomeric reagents in the form of particles of the composition.

In specific embodiments, provided herein is a composition of particulate oligomeric reagents that consist of and/or contain multiple tetramers of streptavidin or streptavidin mutein and that contain multiple binding sites that reversibly bind to one or more reagents, agents, for example, a receptor- binding agents such as anti-CD3 and/or anti-CD28 Fab having streptag. In some embodiments, the particulate oligomeric reagents have a median, median, or median radius between 50 nm and 150 nm; average, median or median molecular weight between 1×10 ⁷ g/mol and 1×10 ⁹ g/mol; and/or an average, median or median amount between 1000 and 5000 streptavidin tetramers or streptavidin mutein. In some embodiments, at least 95% of the particulate oligomeric reagents of the composition have a radius, molecular weight, or number of tetramers that are within ±50%, ±40%, ±30%, ±25%, ±20%, ±15 %, ± 10%, ± 5%, ± 1% of the average, median or median radius, molecular weight or number of tetramers of oligomeric reagents in the form of particles of the composition.

In some embodiments, provided herein is a composition of particulate oligomeric reagents that consist of and/or contain multiple tetramers of streptavidin or streptavidin mutein and that contain multiple binding sites that reversibly bind to one or more agents. In some embodiments, the particulate oligomeric reagents have a median, median, or median radius between 50 nm and 150 nm; average, median or median molecular weight between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol; and/or a mean, median or median amount between 2000 and 4000 streptavidin tetramers or streptavidin mutein. In some embodiments, at least 95% of the particulate oligomeric reagents of the composition have a radius, molecular weight, or tetramer count that is within ±25%, ±20%, ±15%, ±10%, ±5%, ±1 % average, median or median radius, molecular weight or number of tetramers of oligomeric reagents in the form of particles of the composition. In particular embodiments, the particulate oligomeric reagents do not undergo a size increase of greater than 10% when stored at -80° C. for at least 9, 27 weeks, or 46 weeks.

In some embodiments, any of the provided oligomeric reagents are prepared using the method for making or creating oligomeric reagents described in Section II.B below.

B. Preparation of Particulate Oligomeric Reagents

This document provides methods for creating, manufacturing and/or obtaining reagents that consist of oligomerized reagents, i. Oligomeric reagents in the form of particles. In particular embodiments, the particulate oligomeric reagents contain a plurality of binding sites that are capable of reversibly binding to an agent, such as a stimulant. In some embodiments, the particulate oligomeric reagents comprise a plurality of binding sites that are capable of reversibly binding to agents, such as stimulatory agents and/or selective agents, that recognize and/or bind to one or more molecules expressed on the cell. In some embodiments, the methods provided herein are used to create, manufacture and/or obtain oligomeric reagents in the form of particles of the desired or target size.

This document provides methods for obtaining, creating and/or manufacturing reagents that are oligomeric reagents in the form of particles. In some embodiments, the methods provided herein are used to prepare, create, and/or make particulate oligomeric reagents that contain and/or consist of multiple molecules, such as streptavidin tetramers or streptavidin mutein. In some embodiments, the methods provided herein are for the preparation, formulation, and/or manufacture of particulate oligomeric reactants that are soluble reactants. In some embodiments, the methods provided herein for preparing, creating, and/or manufacturing particulate oligomeric reagents include or comprise the step of incubating, processing, and/or contacting molecules, for example, streptavidin tetramers or streptavidin mutein, under conditions suitable for oligomerization. molecules. In some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reactants include the step of separating the particulate oligomeric reactants from non-oligomerized molecules. In some embodiments, the methods provided herein include the step of stabilizing one or more properties of the particulate oligomeric reactants, such as particle size.

In specific embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reactants include a step of oligomerizing molecules, a step of removing oligomerized molecules from non-oligomerized molecules, and/or a step of stabilizing a property of the oligomeric particulate reactants. In some embodiments, the methods provided herein for preparing, creating, and/or making particulate oligomeric reagents comprise and/or include one or more steps of adding a functional group to a molecule, e.g., a functional group that is suitable for a crosslinking or oligomerization reaction. In some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reagents comprise and/or include one or more steps of adding a functional group to a molecule and one or more steps of oligomerizing the molecule. In specific embodiments, the methods provided herein for preparing, creating, and/or manufacturing particulate oligomeric reagents comprise and/or include the steps of adding one or more functional groups to molecules, the step of oligomerizing the molecules, and the step of separating oligomerized molecules, e.g., oligomeric particles, from non-oligomerized molecules.

In some embodiments, molecules that are oligomerized are proteins, polypeptides, peptides, and/or molecules that contain or include one or more amino acids. In some embodiments, a molecule that is oligomerized contains a plurality of binding sites that are capable of binding to an agent, such as a receptor binding agent. In some embodiments, a molecule that is oligomerized contains a plurality of binding sites that are capable of binding to an agent as described in section II(C)(3). In some embodiments, a molecule that is oligomerized contains a plurality of binding sites that are capable of binding to a binding partner, such as binding partner C. In specific embodiments, a molecule that is oligomerized contains a plurality of binding sites that are capable of binding to a binding partner C, which is described in section II(A). In some embodiments, the molecule that is oligomerized is or contains streptavidin, a mutein, or a streptavidin analog, avidin, a mutein, or an avidin analog (eg, neutravidin). In some embodiments, streptavidin is a tetramer in its native state. Thus, in some embodiments, the molecule is a tetramer of streptavidin, mutein, or a streptavidin analog, avidin, mutein, or avidin analog (eg, neutravidin). In specific embodiments, the implementation of the molecule is any of the reagents described in section II(A). In some embodiments, the implementation of the molecule is a tetramer of the reagents described in section II(A).

In certain embodiments, it is contemplated that the characteristics, such as size, of the particulate oligomeric reagents that are made, prepared, and/or created using the methods presented herein depend on the timing of the various steps, techniques, and incubations, as well as on such conditions such as pH and temperature, and reagent concentrations at different steps or stages of the procedure. Thus, in particular embodiments, one or more of the steps or steps of the methods provided herein are performed and/or recorded with precise timing and measurements, for example, to ensure that when the methods provided herein are repeated, the resulting manufactured oligomeric reagents in the form of particles had the same or similar size and characteristics as other batches or lots obtained using the methods presented in this document. For example, in some embodiments, buffer and reagent measurements should be within ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.1%, ±0.01%, or ± 0.001% of the target or desired amount or concentration. In some embodiments, the reaction, e.g., incubation, treatment, or contacting, is carried out at the desired or target pH within pH ± 1, ± 0.5, ± 0.1, ± 0.05, ± 0.04, ± 0, 03, ± 0.02, ± 0.01, ± 0.001, or ± 00001. In specific embodiments, the incubation, treatment or contacting is carried out within 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes , 2 minutes, 90 seconds, 60 seconds, 45 seconds, 30 seconds, 15 seconds, 10 seconds, 5 seconds, or within 1 second of the target or desired time period. In specific embodiments, the time between steps, steps and/or reactions, such as incubations or treatments, is within 30 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 90 seconds, 60 seconds, 45 seconds, 30 seconds, 15 seconds, 10 seconds, 5 seconds, or within 1 second of the set target or desired time.

In some embodiments, certain features of the methods provided herein for making, preparing, or creating particulate oligomeric reagents are critical to the stable production of particulate oligomeric reagents. For example, in some embodiments, the methods provided herein include the step of thiolating the molecules, for example, by incubating the molecules with a thiolating agent, and the time, pH and/or concentrations and amounts of incubation reagents are all reduced within ± 5%, ± 2% , ± 1%, ± 0.1%, ± 0.01%, or ± 0.001% of target or desired values to achieve stable production of oligomeric particulate reagents. In some embodiments, the amount of time between the end of the molecule thiolation step and the molecule oligomerization step, e.g., by incubation of activated and thiolated molecules, falls within ±5%, ±2%, ±1%, ±0.1%, ±0.01 % or ±0.001% of desired or target time period. In some embodiments, the methods provided herein include the step of activating the molecules, for example, by incubating the molecules with an activating agent that adds functionality to the molecules, and the time, pH, and/or concentrations and amounts of incubation reagents are all reduced to within ±5 %, ±2%, ±1%, ±0.1%, ±0.01%, or ±0.001% of target or desired values to achieve stable production of particulate oligomeric reagents. In specific embodiments, the time, pH, and/or concentrations and amounts of reagents for the molecular oligomerization step are all reduced within ±5%, ±2%, ±1%, ±0.1%, ±0.01%, or ±0.001% of target or the desired values to achieve stable production of oligomeric reagents in the form of particles. In specific embodiments, the stable production of particulate oligomeric reagents results in or includes the production of stable lots or batches. Thus, in some embodiments, the methods provided herein result in stable batches or batches of particulate oligomeric reactants. For example, in some embodiments, the methods provided herein result in batches or batches of oligomeric particulate reactants with average, e.g. median, particle sizes that fall within ±50%, ±25%, ±20%, ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, ± 0.01% or ± 0.001% of the average , for example, the median particle size of batches or batches made, obtained or created using the methods in this document.

In some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reagents include the step of activating molecules, e.g., streptavidin tetramers or streptavidin mutein, by incubating, treating, and/or contacting the molecules with an activating agent. In some embodiments, the activating agent adds or is capable of adding a functional group to the molecule that is or is capable of being reactive in a crosslinking reaction. In some embodiments, the activating agent adds or is capable of adding a functional group to one or more amines of the molecule. In some embodiments, the activating agent adds or is capable of adding an amino-reactive group, a sulfhydryl-reactive or thiol-reactive group, an aldehyde-reactive group, a photoreactive group, and/or a hydroxyl-reactive group to the molecule. In some embodiments, the activating agent adds or is capable of adding a sulfhydryl-reactive or thiol-reactive group to the molecule. In some embodiments, the activating agent adds or is capable of adding a haloacetyl group, maleimide group, aziridine group, acryloyl group, arylating agent, vinyl sulfone group, pyridyl disulfide, TNB-thiol, or disulfide reducing agent to the molecule. In some embodiments, the activating agent adds or is capable of adding a maleimide group to the molecule. In some embodiments, the activating agent is or contains sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo SMCC) and/or succinimidyl 6-[(β-maleimidopropionamido)hexanoate (SMPH).

In specific embodiments, molecules, for example streptavidin or streptavidin mutein tetramers, are incubated, treated and/or contacted with an activating agent under conditions suitable for activating the molecule, i.e. One or more functional groups are added to the molecules. In specific embodiments, the implementation of the incubation, processing or contact of the activating agent with molecules is carried out at neutral pH. In some embodiments, the incubation, treatment, or contact of the activating agent with the molecules is carried out at a pH between 5.0 and 9.0, between 6.0 and 8.0, between 6.5 and 7.5, or between 7.0 and 7.5. In some embodiments, the activating agent is incubated, treated, or contacted with molecules at a pH of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1 , about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the pH is about 7.2. In specific embodiments, the pH is 7.2 ± 0.1, ± 0.05, ± 0.02, ± 0.01, ± 0.005, or ± 00001.

In some embodiments, the incubation, processing and/or contacting of the activating agent with molecules, for example streptavidin tetramers or streptavidin mutein, is carried out at a constant temperature. In some embodiments, the incubation, processing and/or contacting of the activating agent with the molecules is carried out at a temperature of at least 4°C, at least 8°C, at least 12°C, at least 16°C, at least at least 20°C, at least 24°C, at least 28°C, at least 32°C, at least 37°C, at least 39°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C or at least 100°C. In specific embodiments, the incubation, processing and/or contact of the activating agent with the molecules is carried out at a temperature between 4°C and 39°C, between 10°C and 37°C, between 10°C and 25°C, between 20°C C and 30°C, between 24°C and 39°C or between 40°C and 100°C. In specific embodiments, the implementation of the incubation, processing and/or contact of the activating agent with the molecules is carried out at room temperature. In some embodiments, the implementation of the incubation and/or processing for oligomerization of molecules is carried out at or approximately at 24°C. In some embodiments, the incubation and/or treatment to activate the molecules is carried out at 24°C ± 2°C, ± 1°C, ± 0.5°C, ± 0.2°C, ± 0.1°C, ± 0 .05°C or ± 0.01°C.

In some embodiments, the incubation, treatment and/or contacting of the activating agent with molecules, for example streptavidin tetramers or streptavidin mutein, is carried out for a period of time. In some embodiments, the incubation, treatment and/or contact of the activating agent with the molecules is carried out for 5 minutes to 1 hour, 15 minutes to 2 hours, 30 minutes to 90 minutes, 1 hour to 6 hours, 6 hours to 24 hours or more than 24 hours. In some embodiments, the incubation, treatment, and/or contact of the activating agent with the molecules is for about 5 minutes, 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, approximately 6 hours, approximately 8 hours, approximately 12 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, or approximately 24 hours. In some embodiments, the incubation, processing and/or contacting of the activating agent with the molecules is carried out for or about 1 hour. In specific embodiments, the incubation, treatment, or contact of the activating agent with the molecules is carried out for 1 hour ± 5 minutes, ± 2 minutes, ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 seconds.

In specific embodiments, the activating agent is incubated, treated and/or contacted with molecules, eg streptavidin or streptavidin mutein tetramers, at a certain molar ratio of activating agent to molecules. In some embodiments, the molar ratio of molecules to activating agent is or is approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In specific embodiments, the molar ratio of molecules to activating agent is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 , ± 10%, ± 5%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, ± 0.05% or ± 0.001%. In some embodiments, the mole ratio is 1:2, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%. In some embodiments, the implementation of the molar ratio is 1:2 ± 5%. In specific embodiments, the implementation of the molar ratio is 1:2 ± 2%.

In some embodiments, incubation, treatment and/or contact with an activating agent and molecules, for example streptavidin or streptavidin mutein tetramers, is completed by removing the activating agent from the molecules. In some embodiments, the implementation of the activating agent is removed from the molecules using chromatography. In some embodiments, the implementation of the activating agent is removed from the molecules using size exclusion chromatography, for example, using a column for desalting.

In specific embodiments, the methods provided herein for the preparation, creation and/or manufacture of particulate oligomeric reagents include the step of thiolating molecules, e.g., streptavidin tetramers or streptavidin mutein, by incubating, treating, and/or contacting the molecules with a thiolating agent. In some embodiments, a thiolating agent is an agent that adds or is capable of adding a thiol functional group to the molecule. In some embodiments, the thiolating agent is an agent that adds or is capable of adding a thiol functional group to one or more free amines. In some embodiments, a thiol functional group is added to the N-terminal amino group and/or to free amines present on the lysine residues of the molecule. In some embodiments, the thiolating agent is or contains a cyclic thioimidate compound. In particular embodiments, the thiolating agent is or contains 2-iminothiolane (Trot's reagent). In some embodiments, the thiolating agent is or contains 2-iminothiolane and adds a thiol functionality to a free amine in a reaction that is illustrated below:

In specific embodiments, the thiolating agent, eg, 2-iminothiolane, is sold, stocked, and/or prepared as a hydrochloride, eg, 2-iminothiolane-HCl salt. Thus, in some embodiments, the addition of 2-iminothiolane to the solution causes a significant drop in the pH of the solution. In some embodiments, the implementation of the solution may be buffered to prevent or reduce the drop in pH. In certain embodiments, it is contemplated that thiolation reactions performed at acidic pH and/or pH below 7.0 limit the availability of lysine residues, e.g., limit the availability of free amines on lysine residues, and thus limit the amount of thiol functionality that adds thiolating reagent molecule. In some embodiments, amino groups on lysine residues can be protonated to an extent that reduces or prevents the ability to thiolate and/or add thiol functional groups to amino groups at acidic pH values and/or at pH values below 7.0. Thus, in some embodiments, the acidity of the solution containing the thiolating agent is adjusted and/or neutralized to increase the efficiency of the thiolation reaction.

In some embodiments, the implementation of the incubation, processing and/or contacting the thiolating agent with molecules includes adding the thiolating agent to a buffer with an alkaline pH or with a pH greater than 7.0 before or at the beginning of incubation, processing, contacting the thiolating agent with molecules. In specific embodiments, a thiolating agent is added to a buffer that has a pH of at least 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least at least 8.0, at least 8.1, at least 8.2, at least 8.3, at least 8.4, at least 8.5, at least 8.6, at least 8.7, at least 8.8, at least 8.9, at least 9.0, at least 9.5, or at least 10.0 before or at the beginning of incubation, treatment, contact of the thiolating agent with molecules . In some embodiments, a thiolating agent is added to a buffer that has a pH of about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2 , approximately 8.3, approximately 8.4, approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, about 9.4 or about 9.5 before or at the beginning of incubation, processing, contact of the thiolating agent with molecules. In some embodiments, the pH of the buffer is about 8.5. In specific embodiments, the pH of the buffer is 8.5 ± 0.1, ± 0.05, ± 0.02, ± 0.01, ± 0.005, or ± 00001. In some embodiments, the buffer contains a buffering agent with a pK _a at room temperature greater than 7.0, greater than 7.5, greater than 8.0, greater than 8.5, or greater than 9.0. In specific embodiments, the buffering agent is or contains TES, HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, tricine, Gly-gly, bicine, HEPBS, TAPS, AMPD, TABS, AMPSO, CHES, CAPSO, AMP, CAPS, CABS and/or borate. In some embodiments, the buffer is or contains a borate buffer. In particular embodiments, the borate buffer contains at least 25 mM borate, at least 50 mM borate, at least 75 mM borate, or about or at least 100 mM borate. In specific embodiments, the buffer is or contains 100 mM ± 10%, ± 5%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, ± 0.05%, or ± 0.001% borate.

In some embodiments, the implementation of the incubation, processing and/or contacting the thiolating agent with molecules is carried out at an alkaline pH or a pH above 7.0. For example, in some embodiments, the pH of the solution upon addition of the thiolating agent and molecules is an alkaline pH or a pH greater than 7.0. In some embodiments, the pH during incubation, treatment, or contact of the thiolating agent with molecules is between 7.0 and 11.0, between 7.0 and 9.0, between 7.5 and 8.5, or between 7.5 and 8.0. In some embodiments, the thiolating agent is incubated, treated, or contacted with molecules at a pH of about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7 .6, about 7.7, about 7.8, about 7.9, or about 8.0. In some embodiments, the pH during incubation, treatment, or contact of the thiolating agent with the molecules is, or is about, 7.7. In specific embodiments, the pH during incubation, processing, or contact of the thiolating agent with molecules is 7.7 ± 0.1, ± 0.05, ± 0.02, ± 0.01, ± 0.005, or ± 00001.

In some embodiments, the incubation, treatment and/or contacting of the activating agent with molecules, for example streptavidin tetramers or streptavidin mutein, is carried out for a period of time. In some embodiments, the incubation, treatment and/or contact of the activating agent with the molecules is carried out for 5 minutes to 1 hour, 15 minutes to 2 hours, 30 minutes to 90 minutes, 1 hour to 6 hours, 6 hours to 24 hours or more than 24 hours. In some embodiments, the incubation, treatment, and/or contact of the activating agent with the molecules is for about 5 minutes, 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, approximately 6 hours, approximately 8 hours, approximately 12 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, or approximately 24 hours. In some embodiments, the incubation, processing and/or contacting of the activating agent with the molecules is carried out for or about 1 hour. In specific embodiments, the incubation, treatment, or contact of the activating agent with the molecules is carried out for 1 hour ± 5 minutes, ± 2 minutes, ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 seconds. In some embodiments, the incubation, processing and/or contacting of the activating agent with the molecules is carried out for or about 25 minutes. In specific embodiments, the incubation, treatment, or contact of the activating agent with the molecules is carried out for 25 minutes ± 5 minutes, ± 2 minutes, ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 seconds.

In certain embodiments, it is contemplated that incubation, processing, and/or contact of a molecule, for example, a streptavidin molecule or a streptavidin mutein, results in a first reaction that adds the desired thiol functionality. However, in some embodiments, the desired thiol functionality may be re-isomerized to a more stable but inactive N-substituted form. Thus, in some embodiments, thiolation of a molecule with 2-iminothiolane adds a thiol functionality to the molecule that can re-isomerize to a more stable N-substituted form without the same reactivity as the thiol functionality (Singh et al. Anal Biochem 236( 1): 114-1125 (1996)). An image of this reaction is shown below:

Therefore, in some embodiments, thiolation of a molecule with 2-iminothiolane should not result in a standard saturation curve and instead will result in a curve where the level or amount of thiol functional groups that are present on the molecules, e.g. streptavidin tetramers or streptavidin mutein, will be reach a peak or maximum level, and then should fall again after reaching the maximum. In some embodiments, the half-life of the thiol functional group is 139 minutes.

In some embodiments, the maximum or peak level of thiol functional groups attached to molecules that is reached during the thiolation reaction is influenced by the pH of the solutions where the reaction takes place. In some embodiments, the maximum or peak level of thiol functionality is greater when the thiolating agent is added to a more alkaline pH buffer than when the thiolating agent is added to a less alkaline buffer. In some embodiments, the maximum or peak level of thiol functionality is reached in a shorter period of time when the thiolating agent is added to a more alkaline pH buffer than when the thiolating agent is added to a less alkaline buffer. In some embodiments, the maximum or peak level of thiol functional groups is greater when the thiolating agent is added to a buffer at or about pH 8.5 than when the thiolating agent is added to a less alkaline buffer, such as a buffer at pH 8.3. In some embodiments, the maximum or peak level of thiol functional groups is reached in a shorter period of time when the thiolating agent is added to a buffer with a pH equal to or approximately equal to 8.5 than when the thiolating agent is added to a less alkaline buffer, for example, a buffer with a pH of 8 ,3. In some embodiments, the maximum or peak level of thiol functional groups is greater when the pH of the solution in which the incubation, treatment, and/or contact with the thiolating agent and molecule during the reaction is at or about pH 7.7 is greater than when the incubation is , processing and/or bringing into contact during the reaction takes place in solution with a pH of less than 7.7, for example, a pH of approximately 6.9. In some embodiments, the maximum or peak level of thiol functional groups is reached in a shorter period of time when the pH of the solution in which the incubation, processing and/or contact with the thiolating agent and the molecule during the reaction takes place is or is approximately pH 7, 7 than when the incubation, handling and/or contacting takes place during the reaction in a solution with a pH of less than 7.7, for example a pH of about 6.9.

In some embodiments, the maximum or peak level of thiol functionality that is added to the molecule is an average (eg, median) that is expressed as the amount of thiol functionality that is added to the molecule. In some embodiments, the maximum or peak level of thiol functional groups is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 20, at least 25, at least 30, at least 40, or at least 50 thiol functional groups. In some embodiments, the maximum or peak level of thiol functionality that is added to each molecule is the average (eg, median) percentage of lysine residues per molecule with an attached or added thiol functionality. In some embodiments, the maximum or peak level is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% , at least 95% or at least 99% lysine residues with an attached or added thiol functional group. In specific embodiments, the molecule is a streptavidin or streptavidin mutein tetramer and the maximum or peak level of thiol functionality is at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 thiol functional groups per tetramer.

In some embodiments, the molecule is incubated, treated, and/or contacted with a thiolating agent for a period of time that is sufficient to achieve the maximum or peak level of thiol functionality that is added to the molecule. In specific embodiments, the maximum or peak level is reached within 1 minute, within 2 minutes, within 5 minutes, within 10 minutes, within 15 minutes, within 20 minutes, within 25 minutes, within 30 minutes, within within 45 minutes, within 60 minutes, within 90 minutes, or within 120 minutes of incubation, treatment, or contact of the thiolating agent with the molecule.

In specific embodiments, a thiolating agent is incubated, treated and/or contacted with a molecule and the amount of thiol functionality that is added to or coupled to the molecules peaks or peaks and then begins to decline after the peak or peak is reached. In some embodiments, the implementation of the incubation, contact and/or processing ends after reaching the peak or maximum level. In some embodiments, the incubation, treatment, or contact is completed at or before the amount of thiol functional groups attached to the molecules is 50% less, 40% less, 30% less, 25% less, 20% less, 15 % less, 10% less, 5% less, or 1% less than the maximum or peak level. In some embodiments, the incubation, processing and/or contacting is completed at the point in time when the average (e.g. median) number of thiol functional groups is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 20, at least 25, at least 30, at least 40 or at least 50 thiol functional groups. In some embodiments, the incubation, contacting and/or treatment is completed at a point in time when at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the lysine residues have an attached or added thiol functional group. In specific embodiments, the molecule is a tetramer of streptavidin or a streptavidin mutein, and incubation, contacting and/or processing is completed at the point in time when the number of thiol functional groups is at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 thiol functional groups per tetramer. In specific embodiments, the implementation of the incubation, processing and/or contacting the thiolating agent and the molecule is completed after 1 hour. In some embodiments, the incubation, processing and/or contacting of the thiolating agent and the molecule is completed after 25 minutes.

In some embodiments, the incubation, processing and/or contacting of the thiolating agent with molecules, for example streptavidin tetramers or streptavidin mutein, is carried out at a constant temperature. In some embodiments, the incubation, processing and/or contacting of the thiolating agent with molecules is carried out at a temperature of at least 4°C, at least 8°C, at least 12°C, at least 16°C, at least at least 20°C, at least 24°C, at least 28°C, at least 32°C, at least 37°C, at least 39°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C or at least 100°C. In specific embodiments, the incubation, processing and/or contacting of the thiolating agent with the molecules is carried out at a temperature between 4°C and 39°C, between 10°C and 37°C, between 10°C and 25°C, between 20°C C and 30°C, between 24°C and 39°C or between 40°C and 100°C. In specific embodiments, the implementation of the incubation, processing and/or contacting the thiolating agent with molecules is carried out at room temperature. In some embodiments, the implementation of the incubation and/or processing for oligomerization of molecules is carried out at or approximately at 24°C. In some embodiments, the incubation and/or treatment for thiolation of molecules is carried out at 24°C ± 2°C, ± 1°C, ± 0.5°C, ± 0.2°C, ± 0.1°C, ± 0 .05°C or ± 0.01°C.

In specific embodiments, the thiolating agent is incubated, treated and/or contacted with molecules, for example streptavidin or streptavidin mutein tetramers, at a certain molar ratio of thiolating agent to molecules. In some embodiments, the incubation, processing and/or contacting of the thiolating agent and molecules is carried out at a molar ratio between 1:1 and 10:1 of thiolating agent and each primary amine per molecule. In specific embodiments, the incubation, treatment and/or contacting of the thiolating agent and molecules is carried out at a 5:1 molar ratio of thiolating agent to each primary amine per molecule. In some embodiments, the molar ratio of thiolating reagent to each primary amine per molecule is, or is approximately, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1. In specific embodiments, the molar ratio of thiolating reagent to each primary amine per molecule is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%. In some embodiments, the mole ratio is 1:5, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%. In some embodiments, the molar ratio of thiolating agent to molecule is between 1:1 and 1000:1, between 1:1 and 500:1, between 10:1 and 200:1, or between 100:1 and 1000:1. In specific embodiments, the implementation of the molar ratio of activating agent to the molecule is approximately 100:1. In some embodiments, the mole ratio is 100:1, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%.

In some embodiments, the incubation, processing and/or contacting of the thiolating agent with molecules, for example streptavidin tetramers or streptavidin mutein, is completed by removing or separating the thiolating agent from the molecules. Methods for removing or separating molecules, for example protein or polypeptide molecules such as streptavidin, are routine in the art and include methods such as chromatography and/or gel filtration. In some embodiments, the thiolating agent is removed from the molecules by chromatography. In some embodiments, the implementation of the activating agent is removed from the molecules using size exclusion chromatography, for example, using a column for desalting.

In some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reactants comprise and/or include the step of oligomerizing the molecules. In some embodiments, molecules are oligomerized by cross-linking single molecules or a complex of subunits that make up a single molecule. In some embodiments, the methods provided herein include one or more steps of treating, incubating, and/or contacting the molecules with an agent that promotes oligomerization. For example, in some embodiments, the molecules are oligomerized by incubating, treating, and/or contacting the molecules with an agent, such as an activating agent, that is a linker or crosslinker, such as a bifunctional linker or crosslinker, or other chemical linker. In some embodiments, the implementation of the linker or cross-linking agent is or contains a bifunctional linker. In some embodiments, the linker is a homobifunctional linker, eg, a linker with at least two of the same functional and/or reactive groups. In specific embodiments, the implementation of the linker is a heterobifunctional linker, for example, a linker with at least two different functional and/or reactive groups. In some embodiments, molecules are incubated with a linker to oligomerize or become oligomerizable. Suitable linkers for the oligomerization of molecules are known in the art and include, but are not limited to, glutaraldehyde, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), dimethyl pimelimidate (DMP), N-hydroxysuccinimide (NHS), dithiobis(succinimidyl propionate (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis[succinimidyl succinate], NHS ester, N-ε-maleimidocaproic acid, N-[ε-maleimidocaproic acid]hydrazide, N-succinimidyl S-acetylthioacetate, N-succinimidyl S-acetylthiopropionate, 2 -iminothiolane (Trot's reagent), 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)-toluene sulfosuccinimidyl, 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate, N-[gamma-maleimidobutyryloxy] sulfosuccinimide ether, N- (K-maleimidundecanoyloxy) sulfosuccinimide ester, maleimidoacetic acid N-hydroxysuccinimide ester, N-(epsilon-maleimidocaproic acid) hydrazide, N-(K-maleimidundecanoic acid) hydrazide, N-(beta-maleimidopropionic acid) hydrazide and hydrazide 3-(2-pyridyldithio)propionyl.

In some embodiments, the methods provided herein comprise and/or involve the oligomerization of molecules that have been modified, eg, chemically modified. In specific embodiments, one or more modified molecules are oligomerized. In specific embodiments, one or more molecules are activated. In some embodiments, the modified molecule is an activated molecule that has been activated by the addition and/or addition of a functional group that is or is capable of being reactive in crosslinking and/or oligomerization reactions. In some embodiments, a functional group is added to or coupled to an amine, such as a primary amine, a molecule, such as an available and/or free amine. In some embodiments, the implementation of the amine, for example, the primary amine is N-terminal amine. In specific embodiments, the implementation of the amine, for example, the primary amine is on the lysine residue. In some embodiments, the activated molecule is modified by adding and/or attaching a functional group that is or contains an amine-reactive group (e.g., N-hydroxysuccinimide ester, imide ester, pentafluorophilic ester, or hydroxymethyl phosphine), sulfhydryl-reactive, or thiol- a reactive group (eg maleimide, haloacetyl, pyridyl disulfide, thiosulfonate or vinyl sulfone), an aldehyde reactive group (eg hydrazide or alkoxyamine), a photoreactive group (eg diazirine or aryl azide), and/or a hydroxyl reactive group (eg isocyanate). In some embodiments, the activated molecule is activated by adding and/or attaching a sulfhydryl-reactive or thiol-reactive group. In some embodiments, the activated molecule is activated by adding and/or attaching a haloacetyl group, maleimide group, aziridine group, acryloyl group, arylating agent, vinyl sulfone group, pyridyl disulfide, TNB-thiol, or disulfide reducing agent. In some embodiments, the activated molecule is activated by adding and/or attaching a maleimide group.

In some embodiments, the modified molecule is a thiolated molecule. In specific embodiments, the modified molecule has been modified by thiolation, such as the addition of a thiol (ie, a thiol group, a thiol function, or a thiol functional group). In specific embodiments, the thiolated molecule is thiolated by addition and/or addition of a thiol functional group with one or more lysine residues.

In some embodiments, the methods provided herein for preparing, creating, and/or making particulate oligomeric reagents comprise and/or include the step of incubating, treating, or contacting activated molecules with thiolated molecules. In some embodiments, incubation, processing and/or contacting oligomerizes and/or results in an oligomerization reaction between thiolated molecules and activated molecules. In specific embodiments, oligomer molecules are formed by incubation, processing and/or contacting thiolated molecules with activated molecules. In some embodiments, the activated molecule has one or more attached maleimide groups. In specific embodiments, the activated molecule is or contains an activated streptavidin or streptavidin mutein molecule. In some embodiments, the activated streptavidin or streptavidin mutein molecule is or contains a streptavidin or streptavidin mutein molecule with one or more attached maleimide groups. In specific embodiments, the thiolated molecule is a thiolated streptavidin or streptavidin mutein molecule. In some embodiments, the thiolated streptavidin or streptavidin mutein molecule is a streptavidin or streptavidin mutein molecule with one or more thiol functional groups. In specific embodiments, the methods provided herein include the step of incubating, contacting and/or treating thiolated streptavidin or streptavidin mutein tetramers with activated streptavidin or streptavidin mutein tetramers, e.g., to oligomerize the streptavidin or streptavidin mutein tetramers.

In specific embodiments, the molecules are oligomerized by a crosslinking reaction. In some embodiments, a portion of the molecules are thiolated by adding one or more thiol functional groups to the molecule. In some embodiments, thiol groups are added to the free amino groups of the molecule, eg, on the amine, eg, primary amine, lysine residue groups and/or the N-terminal amine, eg, the N-terminal primary amine. In some embodiments, a portion of the molecules that are separated from the thiolated molecules are activated by adding or attaching maleimide groups. In some embodiments, the activated molecules do not contain cysteine residues and/or thiol functional groups. As a consequence, in some embodiments, activated molecules do not react with other activated molecules. In some embodiments, the activated and thiolated molecules are incubated and a crosslinking reaction occurs between the maleimide functionality of the activated molecules and the thiol functionality of the thiolated molecules. For example, the crosslinking reaction between a maleimide functional group on the R molecule and a thiol (SH) functional group on the P molecule is illustrated below:

In some embodiments, the reaction between the thiol functional group and the maleimide functional group is suitable for crosslinking molecules to form oligomers. In some embodiments, the molecules are streptavidin or streptavidin mutein tetramers.

In specific embodiments, the molecules, for example, activated and thiolated streptavidin or streptavidin mutein tetramers, are incubated and/or processed under conditions suitable for oligomerization of the molecules. In specific embodiments, the implementation of the incubation and/or processing for oligomerization of molecules is carried out at neutral pH. In some embodiments, the incubation and/or treatment to oligomerize the molecules is carried out at a pH between 5.0 and 9.0, between 6.0 and 8.0, between 6.5 and 7.5, or between 7.0 and 7.5 . In some embodiments, the incubation and/or treatment to oligomerize the molecules is carried out at a pH of about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In some embodiments, the pH is about 7.2. In specific embodiments, the pH is 7.2 ± 0.1, ± 0.05, ± 0.02, ± 0.01, ± 0.005, or ± 00001.

In some embodiments, molecules, such as activated and thiolated streptavidin or streptavidin mutein molecules, are incubated and/or processed under conditions that are suitable for oligomerization of the molecules, and suitable conditions include temperature. In some embodiments, the incubation and/or treatment for oligomerization of molecules is carried out at a temperature of at least 4°C, at least 8°C, at least 12°C, at least 16°C, at least 20°C, at least 24°C, at least 28°C, at least 32°C, at least 37°C, at least 39°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, at least 90°C or at least 100°C. In specific embodiments, the incubation and/or treatment for oligomerization of molecules is carried out at a temperature between 4°C and 39°C, between 10°C and 37°C, between 10°C and 25°C, between 20°C and 30°C , between 24°C and 39°C or between 40°C and 100°C. In specific embodiments, the implementation of the incubation and/or processing for oligomerization of molecules is carried out at room temperature. In some embodiments, the implementation of the incubation and/or processing for oligomerization of molecules is carried out at or approximately at 24°C. In some embodiments, the incubation and/or treatment for oligomerization of molecules is carried out at 24°C ± 2°C, ± 1°C, ± 0.5°C, ± 0.2°C, ± 0.1°C, ± 0 .05°C or ± 0.01°C.

In some embodiments, the molecules, for example, activated and thiolated streptavidin or streptavidin mutein tetramers, are incubated and/or treated under conditions that are suitable to oligomerize the molecules over a period of time. In some embodiments, the molecules are incubated and/or treated under conditions that are suitable for oligomerizing the molecules for 5 minutes to 1 hour, 15 minutes to 2 hours, 30 minutes to 90 minutes, 1 hour to 6 hours, 6 hours to 24 hours or more than 24 hours. In some embodiments, the incubation and/or treatment to oligomerize the molecules is for about 5 minutes, 15 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 3 hours, about 6 hours, approximately 8 hours, approximately 12 hours, approximately 16 hours, approximately 18 hours, approximately 20 hours, or approximately 24 hours. In some embodiments, the implementation of the incubation and/or processing for oligomerization of the molecules is carried out for or about 1 hour. In specific embodiments, the incubation and/or treatment to oligomerize the molecules is carried out for 1 hour ± 5 minutes, ± 2 minutes, ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 second.

In specific embodiments, activated and thiolated molecules, eg, activated and thiolated streptavidin or streptavidin mutein tetramers, are incubated and/or treated to oligomerize the molecules at some molar ratio of activated molecules to thiolated molecules. In specific embodiments, the molar ratio of activated molecules to thiolated molecules is 1:X. In some embodiments, X is an amount, i.e. sum, lysines, and N-terminal amines per thiolated molecule. In some embodiments, X is the number of free or available amino groups per molecule. In some embodiments, X is the number of lysines per thiolated molecule before the addition of thiol functionality. In specific embodiments, the molar ratio of activated molecules to thiolated molecules is or is approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In specific embodiments, the molar ratio of activated molecules to thiolated molecules is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1: 10, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%. In some embodiments, the molar ratio is 1:4, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.1%, ±0.05%, or ±0.001%.

In some embodiments, incubation, treatment and/or contacting under conditions to oligomerize molecules, e.g., streptavidin tetramers or streptavidin mutein, is terminated by the addition of one or more agents, e.g., a chemical agent that stops and/or is able to stop the oligomerization reaction. In some embodiments, the agent is an agent that modifies or otherwise prevents the introduction of one or more functional groups, for example, maleimide or thiol functional groups, into the reaction in a crosslinking or oligomerization reaction. In some embodiments, the molecules are activated and thiolated molecules, and one or more of the agents is or contains an agent that saturates available maleimide groups, such as by adding and/or adding a thiol group, or that cleaves and/or detaches maleimide groups from molecules. In some embodiments, unreacted maleimide groups can be removed with an agent that raises the pH. In some embodiments, the elevated pH will remove and/or detach unreacted maleimide groups while the cross-linked maleimide groups will be stable. In some embodiments, the one or more agents comprise an agent that catalyzes the hydrolysis of the maleimide ring system, for example, via a ring opening reaction. In some embodiments, the molecules are activated and thiolated molecules, and one or more agents is or contains an agent that modifies and/or saturates thiol functional groups. In some embodiments, saturation and/or modification of thiol functional groups prevents oligomerization and/or crosslinking reactions with maleimide groups. In some embodiments, activated and thiolated molecules, such as activated and thiolated streptavidin or streptavidin mutein tetramers, are incubated, treated, and/or contacted with N-ethylmaleimide (NEM) to terminate oligomerization and/or crosslinking reactions.

In some embodiments, the molecules, for example, activated and thiolated streptavidin or streptavidin mutein tetramers, are incubated, treated, and/or contacted with an agent that stops and/or is capable of stopping the oligomerization and/or crosslinking reaction. In some embodiments, an agent that stops and/or is able to stop oligomerization and/or crosslinking reactions is incubated, processed and/or contacted with molecules at a temperature between 4°C and 39°C, between 4°C and 25°C , between 4°C and 10°C or between 20°C and 30°C. In specific embodiments, the implementation of the incubation, processing and/or introduction into contact is first performed at room temperature, and then performed at approximately 4°C. In some embodiments, the implementation of the incubation, processing and/or introduction into contact is first performed at or approximately at 24°C, and then performed at approximately 4°C. In some embodiments, the incubation, treatment, or contact is first performed for about 5 minutes, about 10 minutes, about 15 minutes, about 30 minutes, about 60 minutes, about 90 minutes, or about 120 minutes at room temperature and/or at about 24°C and then incubated, contacted and/or treated for about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 16 hours, about 24 hours or more 24 hours at approximately 4°C. In some embodiments, the incubation, treatment, or contact is first performed for about 15 minutes at room temperature and/or at about 24°C, and then is performed for about 16 hours at about 4°C. In some embodiments, incubation, contact, or contact with NEM is first performed for about 15 minutes at room temperature and/or at about 24°C, and then incubated, contacted, and/or treated for about 16 hours at about 4°C.

In specific embodiments, the methods provided herein for the preparation, creation and/or manufacture of particulate oligomeric reagents include the steps of thiolation of molecules and activation of molecules. In some embodiments, different populations or sets of molecules are thiolated from populations or sets of molecules that are activated. In some embodiments, the activation and thiolation steps are performed at approximately the same time, for example, so that both the thiolated and activated molecules are available for the incubation reaction without the need to store either the thiolated or activated molecules while another process is running. In some embodiments, at least a portion of the incubation, treatment and/or contacting of the thiolating agent with molecules and the incubation, processing and/or contacting of the activating agent with molecules are performed at the same time.

In some embodiments, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 %, at least 70%, at least 80% at least 90% or at least 95% of the incubation, processing and / or contacting of the thiolating agent with molecules is carried out during the incubation, processing and / or contacting activating agent with molecules. In some embodiments, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60 %, at least 70%, at least 80% at least 90% or at least 95% of the incubation, processing and/or contacting of the activating agent with molecules is carried out during the incubation, processing and/or contacting thiolating agent with molecules.

In some embodiments, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 16 hours, or at least 24 hours of incubation, processing and/or administration contacting the activating agent with the molecules is carried out during the incubation, processing and/or contacting the thiolating agent with the molecules. In specific embodiments, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 16 hours, or at least 24 hours of incubation, processing and/or administration the contact of the thiolating agent with the molecules is carried out during the incubation, processing and/or contact of the activating agent with the molecules.

In some embodiments, the incubation, treatment and/or contacting of the thiolating agent and the incubation, treatment and/or contacting of the activating agent with the molecules begin at approximately the same time. In some embodiments, the incubation, treatment and/or contacting of the thiolating agent and the incubation, processing and/or contacting of the activating agent with the molecules begin within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 1 minute, within 30 seconds, within 15 seconds, within 10 seconds, within 5 seconds, within 1 second of each other. In some embodiments, the incubation, treatment and/or contacting of the thiolating agent and the incubation, treatment and/or contacting of the activating agent with the molecules are completed at approximately the same time. In specific embodiments, the incubation, treatment and/or contacting of the thiolating agent and the incubation, processing and/or contacting of the activating agent with molecules are completed within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes, within 1 minute, within 30 seconds, within 15 seconds, within 10 seconds, within 5 seconds, within 1 second of each other.

In certain embodiments, it is contemplated that when a thiol functional group is attached or added to a molecule, the thiol functional group can isomerize to a more stable but inactive N-substituted form. In some aspects, the thiol functionality is added or attached in the presence of 2-iminothiolane (Trot's reagent). In some embodiments, it is contemplated that when a thiol functional group is attached or added to a molecule in the presence of 2-iminothiolane (Trot's reagent), the thiol functional group can isomerize to a more stable but inactive N-substituted form. In some embodiments, the thiol functionality isomerizes with a half-life equal to or approximately equal to 139 minutes after removal of the thiolating agent. Thus, in some embodiments, the number of thiol functional groups on molecules after incubation, treatment and/or contact with a thiolating agent decreases over time.

In specific embodiments, the methods provided herein for the preparation, creation and/or manufacture of particulate oligomeric reagents include the steps of thiolation of molecules, activation of molecules, and oligomerization of molecules, for example, incubation of activated and thiolated molecules under conditions suitable for oligomerization. In specific embodiments, the start of the oligomerization step of the molecule is calculated within or after the exact amount of time after the completion or end of the thiolation step. In some embodiments, the start of the oligomerization step of the molecule is calculated within or after the exact amount of time after the completion or end of the thiolation and activation step.

In specific embodiments, the oligomerization step of the molecule, eg, incubation of the activated and thiolated molecules under conditions suitable for oligomerization, is started within a certain period of time after the end of the thiolation step, eg, after the molecule has been incubated with the thiolating agent. In some embodiments, the oligomerization step of the molecule is started or started before loss, reduction, or decay of 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0, 1%, 0.01%, 0.001% or 0.0001% thiol functional groups that are connected to the molecule at the end of the thiolation step. In specific embodiments, the oligomerization step of the molecule is started or started prior to the loss, reduction, or breakdown of the 10% thiol functionality that is attached to the molecule at the end of the thiolation step. In some embodiments, the oligomerization step of the molecule is started or started within 24 hours, within 16 hours, within 12 hours, within 8 hours, within 6 hours, within 4 hours, within 2 hours, within 90 minutes, within 60 minutes, within 45 minutes, within 30 minutes, within 15 minutes, within 10 minutes, within 5 minutes, or within 1 minute of the end of the thiolation step. In some embodiments, the molecule oligomerization step is started or started after 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, 45 minutes, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute ± 5 minutes, ± 2 minutes, ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 second. In some embodiments, the oligomerization step of the molecule is started or started within 15 minutes of the end of the thiolation step. In specific embodiments, the molecule oligomerization step is started or started 10 minutes ± 1 minute, ± 30 seconds, ± 15 seconds, ± 10 seconds, ± 5 seconds, or ± 1 second after the end of the thiolation step.

In specific embodiments, the end of the thiolation step, eg, incubation of the molecule with the thiolating agent, occurs or occurs when the thiolating agent is removed from the molecules, or when the process for removing the thiolating agent is started or started. In some embodiments, the end of the thiolation step occurs or occurs when the process for removing the thiolating agent is started or started. In specific embodiments, the method for removing the thiolating agent is or includes chromatography, such as size exclusion chromatography. In some embodiments, the end of the thiolation step occurs or occurs when or at the time when the sample or solution containing the thiolating agent and molecules is poured into a chromatographic column, such as a size exclusion chromatography column, to remove or separate the thiolating agent from the molecules. In specific embodiments, the start of the oligomerization step of the molecules is started and/or initiated when the activated molecules are contacted, added and/or mixed with the thiolated molecules.

In specific embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reactants include the step of removing and/or separating particulate oligomeric reactants and/or oligomerized molecules from non-oligomerized molecules. In some embodiments, the step of removing and/or separating particulate oligomeric reagents and/or oligomerized molecules from non-oligomerized molecules occurs after the molecular oligomerization step is completed or terminated.

In some embodiments, oligomerized particulate reactants and/or oligomers of molecules are removed and/or separated from non-oligomerized molecules and from oligomeric particles that are smaller or smaller than a threshold size. In some embodiments, the size threshold is or contains a radius of at least 5 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 25 nm, at least 30 nm, at least at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 75 nm, at least 80 nm, at least 85 nm or at least 90 nm. In some embodiments, the size threshold is or comprises a molecular weight of at least 100 kDa, at least 500 kDa, at least 1000 kDa, at least 2000 kDa, at least 5000 kDa, at least 10000 kDa, at least 50,000 kDa, or at least 100,000 kDa. In some embodiments, the implementation of the threshold size does not affect the average (eg, median) size and/or size distribution of the oligomeric reagents in the form of particles obtained using the methods presented in this document.

In some embodiments, particulate oligomeric reagents, for example, streptavidin tetramer or streptavidin mutein oligomers, are removed and/or separated from non-oligomerized particles by size exclusion chromatography (SEC). In some embodiments, SEC is a technique that separates molecules by size without damaging or destroying the molecules, whereby molecules smaller than the exclusion limit are trapped in the column, and molecules, for example, larger than the exclusion limit, pass through the column without delay. . In some embodiments, the exclusion limit is a size that falls between 1 kDa and 100,000 kDa, between 100 kDa and 10,000 kDa, between 500 kDa and 1000 kDa, between 500 kDa and 5000 kDa, between 5000 kDa and 20,000 kDa, between 10,000 kDa and 50,000 kDa or between 50,000 and 100,000 kDa. In some embodiments, the exclusion limit is greater than the molecular weight of the monomer and/or tetramer of the molecule. In some embodiments, the exclusion limit is greater than the molecular weight of the streptavidin tetramer or streptavidin mutein. In specific embodiments, all particles, eg, oligomeric particles, that pass through the SEC column, eg, headspace, eg, without delay, are collected.

In specific embodiments, when performing SEC, the order in which molecules exit the column is related to the size of the molecules, thus, in some embodiments, the column eluate can be collected in different fractions. In some embodiments, the implementation of the fractions can be combined or discarded to remove particles of certain sizes. For example, in some embodiments, fractions may be discarded to remove particulates, e.g., particulate oligomeric reagents, less than 100 kDa, less than 500 kDa, less than 1000 kDa, less than 2000 kDa, less than 5000 kDa, less than 10,000 kDa, less than 50,000 kDa, or less than 100,000 kDa. In some embodiments, SEC removes particulate oligomeric reactants from non-oligomerized molecules but does not affect the average (eg, median) size and/or size distribution of particulate oligomeric reactants produced by the methods provided herein.

In some embodiments, particulate oligomeric reagents, e.g., oligomerized streptavidin or streptavidin mutein tetramers, can continue to be crosslinked and/or oligomerized after incubation, processing, and/or contacting molecules to complete or terminate oligomerization. Thus, in some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reactants include a stabilization step, such as stabilizing the size of the oligomers. In some embodiments, the step of stabilizing the oligomers comprises or includes incubating, contacting, and/or treating oligomerized molecules, eg, particulate oligomeric reagents, with a stabilizing agent.

In some embodiments, the implementation of the stabilizing agent is any agent that prevents or is able to prevent changes in particle size, for example, oligomeric reagent in the form of particles. In some embodiments, a stabilizing agent is any agent that modifies a functional group on a molecule, or oligomeric species that react or are capable of reacting in an oligomerization and/or crosslinking reaction. In some embodiments, a stabilizing agent is any agent that prevents the formation, isomerization, and/or transformation to obtain a functional group on a molecule, or oligomeric species that react or are capable of reacting in oligomerization and/or crosslinking reactions. In some embodiments, a stabilizing agent is any agent that modifies or is capable of modifying a haloacetyl group, a maleimide group, an aziridine group, an acryloyl group, an arylizing agent, a vinyl sulfone group, a pyridyl disulfide, a TNB-thiol, or a disulfide reducing agent that is attached to the molecule or oligomeric particles. . In specific embodiments, a stabilizing agent is any agent that prevents formation, isomerization, and/or conversion to produce a haloacetyl group, maleimide group, aziridine group, acryloyl group, arylating agent, vinyl sulfone group, pyridyl disulfide, TNB-thiol, or disulfide reducing agent that connected to a molecule or oligomeric particles.

In some embodiments, the stabilizing agent is incubated, processed and/or contacted with the oligomeric particles. In some embodiments, the oligomeric particle is a particulate oligomeric reagent that contains a plurality of thiolated molecules and activated molecules, for example, activated and thiolated streptavidin tetramers or a streptavidin mutein. In some embodiments, the oligomeric particle contains more thiolated molecules that contain an attached N-substituted iminothiolane. In some embodiments, incubation and/or treatment with NEM saturates and/or modifies all available thiol functional groups, thus stopping the crosslinking and/or oligomerization reaction. However, in some embodiments, the N-substituted iminothiolane does not react with NEM and these isomers remain on the molecules and oligomeric particles after incubation with NEM. In some embodiments, re-isomerization of the N-substituted iminothiolane to the thiol isomer may therefore result in post-synthetic growth of the particulate oligomeric reagent, e.g., through additional crosslinking and/or oligomerization reactions, e.g. other oligomeric particles.

In some embodiments, the stabilizing agent is an agent that modifies, removes and/or prevents or is capable of modifying, removing and/or preventing the re-isomerization of the N-substituted iminothiolane to a thiol functional group. In some embodiments, the implementation of the stabilizing agent is or contains hydroxylamine. In some embodiments, an oligomeric particle and/or molecule that contains an attached N-substituted iminothiolane is incubated, treated, and/or contacted with a stabilizing agent that modifies, removes, and/or prevents or is capable of modifying, removing, and/or preventing recurrence. isomerization of N-substituted iminothiolane to a thiol functional group. In specific embodiments, an oligomeric particle and/or molecule that contains an attached N-substituted iminothiolane is incubated, treated and/or contacted with hydroxylamine.

In some embodiments, a stabilizing reagent, eg, hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, eg, particulate oligomeric reagents, eg, to perform a stabilization reaction. In some embodiments, a stabilizing agent, such as hydroxylamine, is added to the oligomerized molecules after the crosslinking reaction between the thiol functional groups and the maleimide functional groups has been carried out and/or completed. In specific embodiments, a stabilizing agent, e.g., hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, e.g., particulate oligomeric reagents, after completion, completion, and/or termination of the crosslinking reaction by adding NEM to the oligomerized molecules. In some embodiments, a stabilizing reagent, e.g., hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, e.g., particulate oligomeric reagents, after completion, completion, and/or termination of the crosslinking reaction by adding NEM to the oligomerized molecules. In some embodiments, a stabilizing reagent is contacted, processed, and/or incubated with oligomerized molecules prior to long-term storage, such as storage at or below room temperature, 4°C, -20°C, or -80°C for at least 1 day, 1 week, 3 weeks, 9 weeks, 27 weeks, 46 weeks, or 1 or more years. In some embodiments, a stabilizing agent, such as hydroxylamine, is contacted, processed, and/or incubated with oligomerized molecules, such as particulate oligomeric reagents, after the oligomerized molecules are loaded onto, passed through, and/or eluted from a column such as as a chromatographic column and/or SEC column. In specific embodiments, a stabilizing reagent is contacted, processed, and/or incubated with oligomerized molecules after the oligomerized molecules are loaded onto, passed through, and/or eluted from the SEC column. In some embodiments, a stabilizing reagent is contacted, processed, and/or incubated with oligomerized molecules to any stage where the oligomerized molecules are loaded onto, passed through, and/or eluted from the SEC column.

In some embodiments, a stabilizing agent, such as hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, such as particulate oligomeric reagents, for, for about, or for at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours or 24 hours. In some embodiments, the stabilizing reagent is contacted, treated, and/or incubated with the oligomerized molecules for between 1 minute and 12 hours, between 1 minute and 1 hour, between 1 minute and 30 minutes, between 5 minutes and 30 minutes, between 10 minutes and 60 minutes, between 10 minutes and 20 minutes, between 1 o'clock and 3 o'clock, between 1 o'clock and 2 o'clock, or between 6 o'clock and 12 o'clock. In specific embodiments, the stabilizing reagent is contacted, treated and/or incubated with the oligomerized molecules for 5 minutes to 30 minutes, or for or about 15 minutes. In some embodiments, the processing, contact and/or incubation is carried out with stirring and/or rocking, for example, gentle rocking and/or stirring.

In some embodiments, a stabilizing agent, such as hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, such as particulate oligomeric reagents, at or near 4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, 37°C, 39°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100° C. In some embodiments, the stabilizing reagent is contacted, processed and/or incubated with oligomerized molecules at a temperature between 4°C and 39°C, between 10°C and 37°C, between 10°C and 25°C, between 20°C C and 30°C, between 24°C and 39°C or between 40°C and 100°C. In specific embodiments, the stabilizing reagent is contacted, processed and/or incubated with the oligomerized molecules at room temperature. In some embodiments, the stabilizing reagent is contacted, processed, and/or incubated with oligomerized molecules at or about 23°C, 24°C, 25°C, or 26°C ± 2°C, ± 1°C, ± 0, 5°C, ± 0.2°C, ± 0.1°C, ± 0.05°C or ± 0.01°C.

In some embodiments, the implementation of the stabilizing reagent, for example, hydroxylamine, removed and/or separated from the oligomerized molecules, for example, oligomeric reagents in the form of particles. In specific embodiments, the stabilization reaction is terminated and/or terminated by separating and/or removing the stabilizing agent from the oligomerized particles. In some embodiments, the implementation of the stabilizing reagent is removed and/or separated from the oligomerized particles using the stage of chromatography. In specific embodiments, the chromatography step is or contains sec. In some embodiments, the implementation of the stabilizing reagent is removed and/or separated from the oligomerized molecules using a column and/or filter. In some embodiments, the column or filter is a desalting column. In some embodiments, the desalting column contains a resin, such as a resin that is or contains Sephadex, dextran, and/or epichlorohydrin.

In specific embodiments, a stabilizing agent, such as hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, such as particulate oligomeric reagents, for 1 minute to 1 hour at a temperature between 4°C and 39°C. , between 10°C and 25°C or between 20°C and 30°C. In some embodiments, a stabilizing agent, such as hydroxylamine, is contacted, treated, and/or incubated with oligomerized molecules, such as particulate oligomeric reagents, for 5 minutes to 30 minutes at a temperature between 10°C and 25°C. .

In particular embodiments, particulate oligomeric reagents, e.g., particulate oligomeric reagents that contain a plurality of streptavidin or streptavidin mutein tetramers that are incubated, treated, or contacted with a stabilizing agent, are size stable. In some embodiments, particulate oligomeric reagents that are incubated, treated, or contacted with a stabilizing agent do not undergo a size change over time of greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater more than 25%, more than 30%, more than 40%, or more than 50%, size changes, such as changes in radius or molecular weight, over a period of time, such as 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, or more than 16 weeks when the particles are stored at room temperature, at or about 4°C or less, at or about -20°C or less, or at or about - 80°C. In some embodiments, particulate oligomeric reagents that are incubated, treated, or contacted with a stabilizing agent do not undergo size increase over time greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater more than 25%, more than 30%, more than 40% or more than 50%, size increase within 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks or more 16 weeks.

In some embodiments, the methods provided herein include one or more steps of filter sterilization of molecules, for example, streptavidin tetramers or streptavidin mutein, and/or particulate oligomeric reagents. In some embodiments, particulate oligomeric reagents are filtered to sterilize. In some embodiments, the particulate molecules or oligomeric reagents are filtered with sterilization before or after incubation with an activating agent or thiolating agent, before or after crosslinking and/or oligomerization of the activated and thiolated molecules, before or after SEC is performed to remove the particulate oligomeric reagents from non-oligomerized molecules, eg tetramers, and/or before or after incubation of oligomeric particulate reagents with a stabilizing agent. In some embodiments, the implementation of the oligomeric reagents in the form of particles filtered with sterilization or allow it. In some embodiments, the particulate oligomeric reagents do not aggregate, clog, or otherwise prevent or interfere with the sterilization filtration process. In some embodiments, sterilization filtration includes passing a solution containing particulate molecules or oligomeric reagents through a porous filter or membrane. In some embodiments, the porous filter or membrane contains pores that are or at least about 0.02 µm, about 0.05 µm, about 0.1 µm, about 0.15 µm, about 0.2 µm, about 0 .22 µm, about 0.3 µm, about 0.4 µm, about 0.45 µm, or about 0.5 µm in diameter. In some embodiments, the pore size is between 0.01 µm and 1.0 µm, between 0.1 µm and.05 µm, between 0.2 µm and 0.25 µm, 0.4 and 0.45 µm, or between 0.2 µm and 0.45 µm. In some embodiments, the implementation of the oligomeric particles have a radius and/or an average radius that is 150 nm or less. In specific embodiments, the implementation of the oligomeric particles have a radius and/or average radius, which is approximately 125 nm, 110 nm or 100 nm or less.

In some embodiments, particulate oligomeric reagents are prepared, created, and/or prepared using the methods provided herein and then stored. In some embodiments, the particulate oligomeric reagents are stored for a period of time prior to any treatments or incubations to combine agents, eg, receptor binding agents, with the particulate oligomeric reagents. In particular embodiments, the particulate oligomeric reagents are stored for a period of time after one or more treatments or incubations to reversibly bind agents, eg, receptor binding agents, to the particulate oligomeric reagents. In some embodiments, particulate oligomeric reagents are stored as two or more aliquots. In some embodiments, particulate oligomeric reagents are stored in a buffer. In some embodiments, the buffer has a neutral pH and/or a pH between 6.5 and 7.5, between 6.8 and 7.4, or about 6.8, about 6.9, about 7.0, about 7.1, about 7.2 or about 7.3. In some embodiments, the particulate oligomeric reagents are stored in a buffer at a pH of approximately 7.2. In some embodiments, the buffer is a phosphate buffer, such as sodium phosphate buffer. In some embodiments, the particulate oligomeric reagents are stored at room temperature, at or about 4°C or less, at or about -20°C or less, or at or about -80°C. In specific embodiments, particulate oligomeric reagents are stored for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks or more than 16 weeks. In some embodiments, the particulate oligomeric reagents are placed in neutral pH buffer and stored at or about -80°C.

In some embodiments, the methods provided herein for preparing, creating, and/or manufacturing particulate oligomeric reagents include or comprise the step of incubating, processing, and/or contacting molecules, for example, streptavidin tetramers or streptavidin mutein, under conditions suitable for oligomerization. molecules, a step of separating oligomeric particulate reactants from non-oligomerized molecules by SEC, and a step of incubating the particles with a stabilizing agent. In some embodiments, the step of incubating, treating and/or contacting the molecules under conditions suitable for oligomerization of the molecules comprises or includes incubating thiolated molecules with one or more attached thiol functional groups with activated molecules with one or more attached maleimide functional groups .

In some embodiments, the methods provided herein for making, creating, and/or making particulate oligomeric reagents include: the step of incubating a plurality of streptavidin or streptavidin mutein tetramers with a thiolating agent in an alkaline pH buffer for a period of 15 to 90 minutes to thiolation of the tetramers, the step of incubating a separate plurality of streptavidin or streptavidin mutein tetramers with an activating agent for a period of time from 30 minutes to 90 minutes to add one or more maleimide functional groups to the tetramers and stop the incubation of 2-iminothiolane and SMPH at the same time, or approximately at the same time, the step of incubating the thiolated and activated tetramers within a certain period of time from five to fifteen minutes after the end of the activation and thiolation incubation, the step of separating the particulate oligomeric reagents from the non-oligomerized molecules using a SEC and a particle incubation step with stabilization to stabilize the particle size of the oligomeric reactants. In some embodiments, the methods include the step of storing the particulate oligomeric reagents in a neutral pH buffer solution at or below -80°C, -20°C, or 4°C.

In some embodiments, the methods provided herein for preparing, creating, and/or making particulate oligomeric reagents include: the step of incubating a plurality of streptavidin or streptavidin mutein tetramers with 2-iminothiolane in an alkaline pH buffer between 7.7 and 8.5 at a temperature approximately 24°C for 60 minutes to thiolate the tetramers, the step of incubating a single plurality of streptavidin or streptavidin mutein tetramers with SMPH at neutral pH 7.2 at a temperature of approximately 24°C for 1 hour to add one or more maleimide functional groups to the tetramers, step stopping the incubation of 2-iminothiolane and SMPH at the same time by separating the incubation of 2-iminothiolane and SMPH from the tetramers by chromatography, e.g. oligomerization reactions between thiolated and activated these tetramers after 60 minutes by incubating the tetramers with NEM, a step of separating oligomeric particulate reactants from non-oligomerized molecules by SEC, and a step of incubating particles with hydroxylamine to stabilize the size of the oligomeric particulate reactants. In some embodiments, the methods include the step of storing the particulate oligomeric reagents in a neutral pH buffer at -80°C. In some embodiments, 2-iminothiolane is added to an alkaline pH 8.5 buffer. In some embodiments, the buffer is or contains 100 mM borate buffer. In some embodiments, the particles are stable, eg, do not undergo a size change greater than 10%, for at least 46 weeks.

In specific embodiments, the methods provided herein for preparing, creating, and/or making particulate oligomeric reagents include: the step of incubating a plurality of streptavidin or streptavidin mutein tetramers with 2-iminothiolane in a buffer with an alkaline pH between 7.7 and 8.5 at a temperature approximately 24°C for 60 minutes for thiolation of tetramers; the step of incubating a single plurality of streptavidin or streptavidin mutein tetramers with SMPH at neutral pH 7.2 at a temperature of approximately 24° C. for 1 hour to add one or more maleimide functional groups to the tetramers; a step of terminating the incubation of 2-iminothiolane and SMPH at the same time by separating the incubation of 2-iminothiolane and SMPH from the tetramers by chromatography, for example, sec; the step of incubating thiolated and activated tetramers, optionally within 10 minutes after the end of the incubation of 2-iminothiolane and SMPH; a step of terminating the oligomerization reaction between the thiolated and activated tetramers after or after about 60 minutes by incubating the tetramers with NEM, a step of incubating the particles with hydroxylamine; a SEC step; and optionally a particle filtration step, for example through a membrane and/or filter with or about a pore size of 0.45 µm and/or 0.2 µm. In some embodiments, the methods include the step of storing the particulate oligomeric reagents in a neutral pH buffer at -80°C. In some embodiments, the particles are stable, eg, undergo a size change of greater than 10% for at least 46 weeks.

C. Reagent Format

1. Media

In some embodiments, the implementation of the reagent is contained on a carrier, such as a solid carrier or surface, such as a bead, or a solid phase or stationary phase (matrix for chromatography). In some such embodiments, the reagent is reversibly immobilized on the support. In some cases, the reagent is immobilized on the carrier through covalent bonds. In some aspects, the reagent is non-covalently reversibly immobilized on a support.

In some embodiments, the carrier is a solid carrier. any solid carrier (surface) can be used for reversible immobilization of the reagent. Illustrative examples of solid supports on which a reagent can be immobilized include a magnetic bead, a polymer bead, a cell culture plate, a microtiter plate, a membrane, an agarose bead, a polystyrene bead, or a hollow fiber. In some aspects, hollow fibers can be used as a bioreactor in the Quantum® Cell Propagation System supplied by TerumoBCT Inc. (Lakewood, CO, USA). In some embodiments, the implementation of the reagent is covalently associated with a solid support. In other embodiments, non-covalent interactions can also be used for immobilization, for example, on plastic substrates. In some embodiments, the reagent may be, for example, streptavidin or an avidin mutein that reversibly binds a streptavidin binding peptide. Such streptavidin muteins can be covalently attached to any surface, such as resin (beads) used for chromatographic purification, and are sold as such by IBA GmbH, Göttingen, such as Strep-Tactin® Sepharose, Strep-Tactin® Superflow®, Strep -Tactin® Superflow® High Capacity or Strep-Tactin® MacroPrep®. Other illustrative examples that are readily available on the market are immobilized metal affinity chromatography (IMAC) resins such as TALON® resins (Westburg, Leusden, Netherlands), which can be used to reversibly immobilize oligo-histidine labeled (his- labeled) proteins, for example, for reversibly binding an agent (eg, a receptor-binding agent or selective agent) that contains as a binding partner C an oligohistidine tag, such as a penta- or hexa-histidine tag. Other examples include calmodulin sepharose available from GE Life Sciences, which can be used in conjunction with an agent (e.g., a receptor binding agent or selective agent) that contains a calmodulin binding peptide as binding partner C or sepharose to which glutathione is bound. In some such cases, the binding partner of C is glutathione-S-transferase.

In some embodiments, the carrier has a solid phase or a stationary phase. Thus, in some embodiments, the reagent is contained on a solid phase or stationary phase (also referred to as a chromatography matrix). In some such embodiments, the reagent is reversibly immobilized on the solid phase or stationary phase. In some cases, the reagent is reversibly immobilized on the stationary phase via covalent bonds. In some aspects, the reagent is non-covalently reversibly immobilized on the stationary phase.

Any material can be used as a matrix for chromatography. Typically, a suitable chromatographic material is essentially harmless, ie. Under the right conditions, it does not damage cell viability, for example, when used in a packed chromatographic column. In some embodiments, the implementation of the stationary phase remains in a given location, for example, in a given position, while the position of the sample is changed. Thus, in some embodiments, the stationary phase is part of the chromatographic system through which the mobile phase flows (either as a through stream or in a batch mode), and where the distribution between the phases of the components contained in the liquid phase (either dissolved or dispersed ).

In some embodiments, the chromatography matrix is in the form of a solid or semi-solid phase, while the sample that contains the target cell to be isolated/separated is in the fluid phase. The chromatography matrix may be a particulate material (of any suitable size and shape) or a monolithic chromatographic material including a paper support or membrane. Thus, in some aspects, chromatography can be both column chromatography and planar chromatography. In some embodiments, columns providing bi-directional flow, such as PhyTip® columns available from PhyNexus, Inc., can be used in addition to standard column/flow-through column chromatography columns. San Jose, CA, U.S.A., or pipette tips. Thus, in some cases, the chromatographic columns used in the present methods are also pipette tips or bidirectional flow columns. In some cases, for example, when a particulate matrix material is used, the particulate matrix material may, for example, have a median particle size of from about 5 µm to about 200 µm, or from about 5 µm to about 400 µm, or from about 5 µm to about 600 µm. In some aspects, the matrix for chromatography may, for example, be or contain a polymer resin or a metal oxide or metalloid oxide. In some aspects, for example, when using planar chromatography, the matrix material can be any material suitable for planar chromatography, such as conventional cellulose-based or organic polymer-based membranes (for example, paper membrane, nitrocellulose membrane, or polyvinylidene difluoride (PVDF) membrane) or silica-coated glass plates. In one embodiment, the chromatography matrix/stationary phase is a non-magnetic material or a non-magnetizable material.

In some embodiments, non-magnetic or non-magnetizable chromatography stationary or solid phases that are suitable for the present methods include derivatizing silica or a cross-linked gel. In some aspects, the crosslinked gel may be based on a natural polymer, such as a class of polymers that occur naturally. For example, a natural polymer on which the stationary phase for chromatography can be based is a polysaccharide. In some embodiments, the solid phase or stationary phase is a polystyrene bead. In some cases, the corresponding polysaccharide is usually crosslinked. An example of a polysaccharide matrix includes, but is not limited to, an agarose gel (eg, Superflow® agarose or Sepharose® material such as Superflow® Sepharose®, which are sold in various bead and pore sizes) or cross-linked dextran(s) gel. A further illustrative example is a cross-linked particulate agarose matrix to which dextran is covalently bonded, sold (in various bead sizes and pore sizes) as Sephadex® or Superdex®, both supplied by GE Healthcare. Another illustrative example of such a chromatographic material is Sephacryl®, which is also sold by GE Healthcare in various bead and pore sizes. Another illustrative example of such a chromatographic material is CytoSorb polystyrene beads.

In some embodiments, the cross-linked gel may also be based on a synthetic polymer, such as a class of polymers that do not occur naturally. In some aspects, the synthetic polymer on which the stationary or solid phase for chromatography is based is a polymer that has polar monomer units and is therefore itself polar. Thus, in some cases, such a polar polymer is hydrophilic. Hydrophilic molecules, also referred to as lipophobic, in some aspects contain moieties that can form a dipole-dipole interaction with water molecules. In general, hydrophobic molecules, also called lipophilic, tend to separate from water.

Illustrative examples of suitable synthetic polymers are polyacrylamide(s), styrene-divinylbenzene gel, and an acrylate-diol or acrylamide-diol copolymer. An illustrative example is the polymethacrylate gel sold as Fractogel®. A further example is the ethylene glycol methacrylate copolymer sold as Toyopearl®. In some embodiments, the chromatographic stationary phase may also contain natural and synthetic polymeric components, such as a composite matrix or a polysaccharide-agarose composite or copolymer, such as a polyacrylamide/agarose composite, or a polysaccharide and N,N'-methylenebisacrylamide. An illustrative example of a dextran-N,N'-methylenebisacrylamide copolymer is the aforementioned Sephacryl® material series. In some embodiments, the derivatized silica may comprise silica particles that are bonded to a synthetic or natural polymer. Examples of such embodiments include, but are not limited to, polysaccharide grafted silica, polyvinylpyrrolidone grafted silica, polyethylene oxide grafted silica, poly(2-hydroxyethylaspartamide) grafted silica, and poly(N-isopropylacrylamide) grafted silica.

In some embodiments, the chromatography matrix is a gel filtration matrix, such as when used in a removal cartridge as described herein. Typically, gel filtration can be characterized by the property it is expected to need. Therefore, the gel filtration matrix, in some aspects, allows cells or other biological entities to be separated primarily on the basis of their size. In some such aspects, the appropriate chromatography matrix is typically the porous particulate material as mentioned above. The matrix for chromatography may have a certain exclusion limit, which is usually defined in terms of molecular weight, above which the entry of molecules into the pores is completely excluded. In some embodiments, the appropriate molecular weight defining the exclusion limit can be selected to be below the weight corresponding to the weight of the target cells. In such an embodiment, the target cell is prevented from entering the pores of the size exclusion chromatography matrix. Also, the stationary phase may have pores that are smaller than the size of the selected target cell. In illustrative embodiments, the implementation of the matrix for chromatography has a median pore size from 0 to about 500 nm.

In some embodiments, components present in the sample, such as agents (e.g., receptor binding agents or selective agents) or a competitive reagent, may have a size lower than the pore exclusion limit and thus may enter the pores of the matrix to chromatography. In some aspects, from those components that are able to partially or completely enter the pore volume, larger molecules with less access to the pore volume may be eluted first, while the smallest molecules are usually eluted last. In some embodiments, the exclusion limit of the chromatography matrix is chosen to be below the maximum width of the target cell. Therefore, in some aspects, components that have access to the pore volume may remain in/on the chromatography matrix longer than the target cell. Thus, in some cases, target cells can be collected in the chromatographic column eluate separately from other sample materials/components. As a consequence, in some aspects, components such as an agent (eg, receptor binding agent or selective agent) or, when applicable, a competitive reagent, may elute from the gel filtration matrix at a later time point than the target cell. In some embodiments, this effect can be further enhanced, for example, if the gel permeation chromatography matrix contains a reagent (e.g., covalently bound thereon) that contains Z binding sites that are capable of binding agents (e.g., receptor binding agents or selective agents ) and/or a competitive reagent present in the sample. In some instances, an agent (eg, a receptor binding agent or a selective agent) and/or a competitive reagent may be bound by the Z binding sites of the reagent and thus immobilized on the matrix. In some aspects, this method is carried out in a removal cartridge.

In some embodiments, the chromatography matrix used in the present methods may also contain a magnetically attracted substance, such as one or more magnetically attracted particles or a ferrofluid. Suitable magnetically attracted particles may contain a reagent with a binding site that is capable of binding to the target cell. In some instances, the magnetically attracted particles may comprise a diamagnetic, ferromagnetic, paramagnetic, or superparamagnetic material. In general, a superparamagnetic material responds to a magnetic field with an induced magnetic field without the resulting permanent magnetization. Iron oxide based magnetic particles are for example purchased from Dynal Biotech as Dynabeads®, from Miltenyi Biotec as Magnetic MicroBeads, from CPG Inc. As magnetic porous glass beads, and various other sources such as Roche Applied Science, BIOCLON, BioSource International Inc. micromod, AMBION, Merck, Bangs Laboratories, Polysciences, or Novagen Inc., just to name a few. Magnetic nanoparticles based on superparamagnetic Co and FeCo as well as ferromagnetic Co nanocrystals have been described, for example, by Hutten, A. et al. (J. Biotech. (2004), 112, 47-63). In other embodiments, the chromatography matrix used in the present methods is devoid of any magnetically attracted material.

In some embodiments, an apparatus is provided that includes at least one arrangement of the first and second stationary phase, such as a chromatographic column for cell selection (sorting cartridge) and a second chromatographic column (removal cartridge) for removing reagents. The device may contain a variety of layouts in which the first and second stationary phases (chromatographic columns) are connected in series with the possibility of passing the fluid. The device may comprise a sample inlet fluidly connected to the first stationary phase of the first arrangement of the first and second stationary phases. In some embodiments, the apparatus may also comprise a cell sample outlet, wherein the sample outlet is fluidly coupled to the second stationary phase of the last of the at least one arrangement of the first and second chromatography stationary phases. In some aspects, the apparatus may also include a competitive reagent container that is fluidly coupled to at least one of the first stationary phases of the first and second stationary phase arrangements.

2. Soluble reagents

In some embodiments, the implementation of the reagent is not associated with a solid carrier, i. It is present in soluble form or is soluble. In principle, the same reagent can be used as in the case of a reagent that is immobilized on a support such as a solid support or a stationary phase. For example, any of the exemplary reagents described above may be used without immobilizing or attaching such a reagent to a carrier, such as without attaching a solid carrier or stationary phase. In some embodiments, the reagent contains a plurality of binding sites, Z, for reversibly binding to the binding agent through interaction with a binding partner, C. molecule (eg, oligomers or polymers of a dimeric, trimeric, or tetrameric protein). In some embodiments, the reagent may be, for example, a streptavidin mutein oligomer, a calmodulin oligomer, a compound (oligomer) that provides at least two K chelating groups, wherein the at least two chelating groups are capable of binding to a transition metal ion, thereby making a reagent capable of binding to an oligohistidine affinity tag, a multimeric glutathione-S-transferase, or a biotinylated carrier protein.

In some embodiments, the implementation of the reagent is characterized by the absence of a solid carrier (surface) associated with the reagent. For example, in some embodiments, the reagent does not contain or is not attached (directly or indirectly) to a particle, bead, nanoparticle, microsphere, or other solid carrier. In some embodiments, the reagent is not rigid, inflexible, or durable, or does not contain or is not bonded to a rigid, inflexible, or durable surface. In some embodiments, the reagent is flexible or substantially flexible. In some cases, the reagent is able to conform or adapt to the shape of the cell surface. In some embodiments, the reagent does not have or contain a shape that is spherical or substantially spherical.

In some embodiments, substantially all of the reagent, i. more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, is, consists of or contains organic material. For example, in some embodiments, more than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the reactant is, consists of or contains lipids, carbohydrates, proteins, peptides, or mixtures thereof. In some embodiments, the reactant is substantially free of, consists of, or contains no inorganic material, an inorganic core, such as a metal core, such as iron, synthetic or inorganic polymers, such as styrenic polymers, such as polystyrene, latex, magnetic cores, or from silicon dioxide. For example, in some embodiments, the implementation of the relative percentage of inorganic material of the reagent or material that is part of the reagent, less than 20%, 15%, 10%, 5% or less.

In some embodiments, a majority (i.e., greater than 50%), such as greater than 60%, 70%, 80%, 90%, 95%, 99%, or more, of the total reagent volume in aqueous solution consists of individual protein molecules that contain a reactant, such as oligomers or polymers of single molecules or a complex of subunits that make up a single molecule (eg, a tetrameric molecule). In some embodiments, the total density of the soluble reactant is less than 1.2 g/cm ³ , 1.1 g/cm ³ , 1.0 g/cm ³ or less.

In some embodiments, the soluble reagent, e.g., not coupled to a carrier or solid carrier (e.g., not coupled to a bead), has a relatively small size, such as a size typically less than or about less than 20 nm, such as less than or about less than less than 15 nm, less than or approximately less than 10 nm, less than or approximately less than 5 nm, or less.

In some embodiments, the soluble reagent, eg, not associated with a carrier or solid carrier (eg, not associated with a bead), is biologically inert, ie. Non-toxic to living cells. In some embodiments, the reagent may be biodegradable, for example, it may be degraded by enzymatic activity or cleaved by phagocytic cells.

In some embodiments, a reagent (eg, streptavidin or a mutein, eg, tetrameric streptavidin muteins) can be reacted with a carrier, such as an organic carrier. In some aspects, in addition to the reaction with the polysaccharide, physiologically or pharmaceutically acceptable proteins such as serum albumin (eg, human serum albumin (HSA) or bovine serum albumin (BSA)) can also be used as the carrier protein. In such cases, a reagent such as streptavidin or a streptavidin mutein (either as a single tetramer or also as oligomers) can be coupled to the carrier protein via a non-covalent interaction. In some such embodiments, biotinylated BSA (which is sold by various vendors such as ThermoFisher Scientific, Sigma Aldrich, or Vectorlabs, to name but a few) can be reacted with a reagent (eg, streptavidin mutein). In some aspects, some of the reagent oligomers (e.g., streptavidin oligomers) can non-covalently bind via one or more Z binding sites to the biotinylated carrier protein, leaving most of the Z binding sites of the oligomer available to the binding agent (e.g., receptor binding agent or selective agent). ) and any additional agent that is described in this document. Thus, using this approach, it is possible to obtain a soluble reagent with multiple Z binding sites.

In some embodiments, a reagent such as a streptavidin mutein (either as a separate tetramer or also as an oligomer) can be covalently linked to a synthetic carrier such as a polyethylene glycol (PEG) molecule. Any suitable PEG molecule can be used for this purpose, for example, and the PEG molecule and the corresponding reagent can be soluble. Typically, PEG molecules up to a molecular weight of 1000 Da are soluble in water or culture medium, which can be used in the present methods. In some cases, such a PEG-based reagent can be prepared using commercially available activated PEG molecules (e.g., PEG-NHS derivatives supplied by NOF North America Corporation, Irvine, California, USA, or activated PEG derivatives supplied by Creative PEGWorks, Chapel Hills, North Carolina , USA) with amino groups of the streptavidin mutein.

3. Agents

In some embodiments, the agent (eg, a receptor binding agent or selective agent) has one or more binding sites, B, for binding to a cell surface molecule, eg, a cell surface molecule. Thus, in some instances, an agent (e.g., a receptor binding agent or selective agent) contains a B binding site or a plurality of B binding sites, where the specific binding between the agent (the receptor binding agent or selective agent) and the molecule on the surface of the target cells involves the interaction between B and the molecule. In some embodiments, the agent contains only one binding site, ie. is monovalent. In some embodiments, the agent (eg, receptor binding agent or selective agent) has at least two, eg, a plurality of B binding sites, including three, four, or five B binding sites capable of binding to a cell surface molecule. In some such aspects, at least two or a plurality of B binding sites may be identical. In some embodiments, one or more of the at least two or more B binding sites may be different (eg, B1 and B2).

In some embodiments, one or more different agents (eg, one or more different receptor-binding agents, selective agents, or other agents that bind to a molecule on a cell) are reversibly linked to a reagent. In some embodiments, the reagent is a particulate oligomeric reagent. In some embodiments, at least 2, 3, 4 or more different agents are reversibly linked to the same reagent. In some embodiments, at least two different agents are reversibly linked to the same reactant, whereby each reactant contains a B binding site or a plurality of B binding sites for specific binding between the agent and the molecule. In some embodiments, at least two or more agents contain the same binding site B, for example, to bind the same or substantially the same molecule. In some embodiments, at least two or more agents contain different B binding sites, for example, to bind to different molecules. In some embodiments, the first agent (e.g., the first receptor binding agent or the first selective agent) contains a B1, B2, B3, B4 binding site, etc., and the second agent (e.g., the second receptor binding agent or the second selective agent ) contains another B1, B2, B3, B4 binding site, etc. In some embodiments, the first agent (eg, the first selective agent) contains a B1 binding site, and the second agent (eg, the second selective agent) contains a B3 binding site. In some embodiments, the first agent (eg, the first receptor binding agent) contains a B2 binding site, and the second agent (eg, the second receptor binding agent) contains a B4 binding site. In any of such embodiments, the first agent and the second agent may comprise a binding partner, C1 or C2. In some embodiments, C1 and C2 may be the same. In some embodiments, C1 and C2 are different. In some embodiments, the first agent and the second agent contain the same binding partner, C1.

In some instances, the dissociation constant (K _d ) of the binding between the agent (for example, via the B binding site) and the Z binding site of the reagent may range from about 10 ^-2 M to about 10 ^-13 M, or from about 10 ^-3 M to about 10 ^-12 M, or from about 10 ^-4 M to about 10 ^-11 M, or from about 10 ^-5 M to about 10 ^-10 M. In some embodiments, the dissociation constant (K _d ) for binding between the binding agent and the molecule has low affinity, for example, in the range of K _d of about 10 ⁻³ to about 10 ⁻⁷ M. In some embodiments, the dissociation constant (K _d ) for binding between the binding agent and the molecule is of high affinity, for example, in the range of K _d from approximately 10 ⁻⁷ to approximately 1×10 ⁻¹⁰ M.

In some embodiments, the dissociation of the binding of the agent through the binding site B and the molecule is sufficiently rapid, for example, providing only temporary staining of the target cell, or binding to the agent after the reversible bond between the reagent and the agent is broken. In some cases, when expressed in terms of speed k_off(also called the dissociation rate constant for binding between the agent (via the binding site B) and the molecule the rate k_offis approximately 0.5×10⁻⁴sec⁻¹or more, approximately 1×10⁻⁴sec⁻¹or more, approximately 2×10⁻⁴sec⁻¹or more, approximately 3×10⁻⁴sec⁻¹or more, approximately 4×10⁻⁴sec⁻¹or more, approximately 5×10⁻⁴sec⁻¹or more, approximately 1×10⁻³sec⁻¹or more, approximately 1.5×10⁻³sec⁻¹or more, approximately 2×10⁻³sec⁻¹or more, approximately 3×10⁻³sec⁻¹or more, approximately 4×10⁻³sec⁻¹, approximately 5×10⁻³sec⁻¹or more, approximately 1×10⁻²SEC or more or approximately 5×10⁻¹sec−1 or more. The skill level of a specialist makes it possible to empirically determine the range of speed k_off, suitable for the interaction of specific agent molecules and cells (see, for example, US Published Application No. US2014/0295458). For example, you can use an agent with a fairly high speed k_off, for example, greater than 4.0×10⁻⁴sec⁻¹, so that after disruption of the binding of the complexes, most of the agent can be removed or dissociated within one hour. In other cases, you can use an agent with a lower rate k_off, for example, 1.0×10⁻⁴sec⁻¹, so that after disruption of the binding of the complexes, most of the agent can be removed or dissociated from the cell within about 3 and a half hours.

In some embodiments, the K _d of this bond, as well as the rate of K _d , k _off , and k _on of the bond formed between the B site of the binding agent (e.g., receptor binding agent or selective agent) and the cell surface molecule, can be determined using any suitable means, for example using fluorescent titration, equilibrium dialysis or surface plasmon resonance.

In some aspects, the cell surface molecule is a molecule against which an agent (eg, a receptor binding agent or a selective agent) can be directed. In some embodiments, the cell surface molecule is a peptide or protein, such as a receptor, eg, a membrane receptor protein. In some embodiments, the implementation of the receptor is a lipid, polysaccharide or nucleic acid. In some embodiments, the cell surface molecule that is the protein may be a peripheral membrane protein or an integral membrane protein. In some embodiments, the implementation of the cell surface molecule may have one or more domains that span the membrane. As a few illustrative examples, a membrane protein with a transmembrane domain can be a G-protein coupled receptor such as odor receptors, rhodopsin-like receptor, pheromone and rhodopsin receptor, peptide hormone receptor, taste receptor, GABA receptor, opiate receptor, serotonin receptor, Ca2+ receptor, melanopsin , a neurotransmitter receptor such as a ligand-gated, voltage-gated, or mechanically-gated receptor, including acetylcholine, nicotinic, adrenergic, norepinephrine, catecholamine, L-DOPA-, dopamine, and serotonin (biogenic amine, endofrin/enkephalin) neuropeptide receptor, receptor kinase such as serine/threonine kinase, tyrosine kinase, porin channel such as chloride channel, potassium channel, sodium channel, OMP protein, ABC transporter (ATP binding cassette transporter) such as amino acid transporter, Na-glucose transporter, Na/iodide transporter , an ion transporter such as luminosity a reactive complex, cytochrome c oxidase, ATPase Na/K, H/K, Ca, a cell adhesion receptor such as a metalloprotease, integrin, or caterin.

In some embodiments, the cell surface molecule may be an antigen that defines a desired population or subpopulation of cells, e.g., a population or subset of blood cells, e.g., lymphocytes (e.g., T cells, T helpers, e.g., CD4+ T helpers, B cells or natural killer cells), monocytes or stem cells, eg CD34 positive peripheral stem cells or Nanog or Oct-4 expressing stem cells. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Treg). An illustrative example of Tregs are CD4 CD25 CD45RA Tregs, and an illustrative example of memory T cells are CD62L CD8+ specific central memory T cells. The cell surface molecule can also be a marker for a tumor cell.

As described above, in some embodiments, the agent (eg, a receptor binding agent or selective agent) has a binding partner C in addition to a B binding site that is capable of connecting a cell surface molecule. In some aspects, a given binding partner C is capable of binding to the Z binding site of a reagent, wherein the reagent has one or more binding sites for the binding partner C. In some embodiments, a non-covalent bond that can be formed between a binding partner C that is contained in an agent (e.g., a receptor binding agent or selective agent) and the Z binding site(s) of the reagent can have any of the desired strength and affinity and may be ruptured or reversible under the conditions under which the method is performed. The agent (e.g., receptor binding agent or selective agent) may contain at least one, including two, three, or more additional C binding partners, and the reagent may contain at least two, e.g., three, four, five, six, seven, eight or more Z attachment sites for the binding partner C that is contained in the agent (eg, receptor binding agent or selective agent). As described in US Pat. No. 7,776,562, US Pat. No. 8,298,782 or International Patent Application WO 2002/054065, any combination of binding partner C and a reagent with one or more corresponding Z binding sites can be selected, for example such that binding partner C and the Z binding site were able to reversibly bind in the complex, for example, to cause an avidity effect.

The binding partner C contained in the agent (eg, receptor binding agent or selective agent), for example, may be hydrocarbon based (including polymeric) and contain nitrogen, phosphorus, sulfur, carbene, halogen or pseudohalogen groups. In some aspects, it may be an alcohol, an organic acid, an inorganic acid, an amine, a phosphine, a thiol, a disulfide, an alkane, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a nucleic acid, a lipid, a saccharide, an oligosaccharide, or a polysaccharide. As further examples, it can also be a cation, an anion, a polycation, a polyanion, a polycation, an electrolyte, a polyelectrolyte, a carbon nanotube, or a carbon nanofoam. Typically, such a binding partner C has a higher affinity for the binding site of the reagent than for another substance. Examples of a suitable binding partner C include, but are not limited to, crown ether, an immunoglobulin, a fragment thereof, and a protein binding molecule with antibody-like functions.

In some embodiments, the binding partner C that is contained in the agent (eg, receptor binding agent or selective agent) contains biotin, and the reagent contains a streptavidin analog or an avidin analog that binds reversibly to biotin. In some embodiments, the binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent) contains a biotin analog that reversibly binds to streptavidin or avidin, and the reagent contains streptavidin, avidin, a streptavidin analog, or an avidin analog, which reversibly binds to the corresponding biotin analogue. In some embodiments, the binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent) contains streptavidin or an avidin binding peptide, and the reagent contains streptavidin, avidin, a streptavidin analog, or an avidin analog that binds reversibly to the appropriate streptavidin or avidin-binding peptide.

In some embodiments, the reagent is a streptavidin, such as a streptavidin mutein, including any of those described above (e.g., those listed in SEQ ID NOs: 3-6 or 60-61) and the binding partner C that is contained in the agent (e.g., receptor binding agent or selective agent) may contain a streptavidin binding peptide. In some embodiments, the streptavidin-binding peptide may contain a sequence with the general formula given in SEQ ID NO: 9, for example, contains the sequence given in SEQ ID NO: 10. In some embodiments, the peptide sequence has the general formula given in SEQ ID NO: NO: 11, such as shown in SEQ ID NO: 12. In one example, the peptide sequence is Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (also called Strep-tag®, shown in SEQ ID NO: 7 ). In one example, the peptide sequence is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (also referred to as Strep-tag® II, shown in SEQ ID NO: 8). In some embodiments, the implementation of the peptide ligand contains a sequential arrangement of at least two streptavidin-binding modules, while the distance between the two modules is at least 0 and not more than 50 amino acids, while one binding module has 3-8 amino acids and contains at least least the sequence of His-Pro-Xaa (SEQ ID NO: 9), where Xaa is glutamine, asparagine or methionine, and while the other binding module has the same or a different streptavidin peptide ligand, such as shown in SEQ ID NO: 11 (See, for example, PCT International Published Application No. WO02/077018; US Patent No. 7981632). In some embodiments, the peptide ligand comprises a sequence having the formula given in any of SEQ ID NOs: 13 or 14. In some embodiments, the peptide ligand has an amino acid sequence given in any of SEQ ID NOs: 15-19.

In some embodiment, the binding partner C of the agent (eg, receptor binding agent or selective agent) contains a moiety known to those skilled in the art as an affinity tag. In such an embodiment, the reagent may contain an appropriate binding partner, for example, an antibody or antibody fragment known to bind to an affinity tag. As a few illustrative examples of known affinity labels, the binding partner C that is contained in the agent (e.g., receptor binding agent or selective agent) may contain dinitrophenol or digoxigenin, oligohistidine, polyhistidine, an immunoglobulin domain, maltose-binding protein, glutathione-S-transferase (GST), chitin-binding protein (CBP) or thioredoxin, calmodulin-binding peptide (CBP), FLAG'-peptide, HA-tag (sequence: Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala ) (SEQ ID NO: 20), VSV-G-tag (sequence: Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys) (SEQ ID NO: 21), HSV-tag (sequence: Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp) (SEQ ID NO: 22), T7 epitope (Ala-Ser-Met-Thr-Gly-Gly-Gln -Gln-Met-Gly) (SEQ ID NO: 22), maltose-binding protein (MBP), HSV epitope of the sequence Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp (SEQ ID NO : 24) glycoprotein D of the herpes simplex virus, epitope "myc" of the transcription factor c-myc of the sequence Gl u-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu (SEQ ID NO: 25), V5-tag (Sequence: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu -Gly-Leu-Asp-Ser-Thr) (SEQ ID NO: 26) or glutathione-S-transferase (GST). In such embodiments, a complex formed between one or more Z binding sites of a reagent, which may be an antibody or antibody fragment, and an antigen can be competitively disrupted by the addition of free antigen, i.e. free peptide (epitope tag) or free protein (such as MBP or CBP). In some embodiments, the affinity tag can also be an oligonucleotide tag. In some cases, such an oligonucleotide label can be used, for example, for hybridization with an oligonucleotide with a complementary sequence associated with or contained in a reagent.

Additional examples of suitable binding partner C include, but are not limited to, lectin, protein A, protein G, metal, metal ion, nitrile triacetic acid (NTA) derivatives, RGD motifs, dextran, polyethyleneimine (PEI), redox polymer, glycoproteins , aptamers, dye, amylose, maltose, cellulose, chitin, glutathione, calmodulin, gelatin, polymyxin, heparin, NAD, NADP, lysine, arginine, benzamidine, poly U, or oligo-dT. Lectins such as concavalin are known to bind to polysaccharides and glycosylated proteins. An illustrative example of a dye is a triazine dye such as Cibacron blue F3G-A (CB) or Red HE-3B, which specifically bind NADH-dependent enzymes. Normally, Green A binds to Co A proteins, human serum albumin and dehydrogenases. In some cases, the dyes 7-aminoactinomycin D and 4',6-diamidine-2-phenylindole bind to DNA. Typically, metal cations such as Ni, Cd, Zn, Co or Cu are commonly used to bind affinity tags such as an oligohistidine containing sequence containing a hexahistidine tag or a His-Asn-His-Arg-His-Lys-His-Gly- tag. Gly-Gly-Cys (MAT tag) (SEQ ID NO: 35) and N-methacryloyl-(L)-cysteine methyl ester.

In some embodiments, the binding between the binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent) and one or more Z binding sites of the reagent occurs in the presence of a divalent, trivalent, or tetravalent cation. In this regard, in some embodiments, the implementation of the reagent contains a divalent, trivalent or tetravalent cation, usually retained, for example, in the form of a complex, through a suitable chelator. In some embodiments, the binding partner C that is contained in the agent (eg, receptor binding agent or selective agent) may contain a fragment that contains, for example, in the form of complexes, a divalent, trivalent or tetravalent cation. Examples of a suitable metal chelator include, but are not limited to, ethylenediamine, ethylenediaminetetraacetic acid (EDTA), ethylene glycoltetraacetic acid (EGTA), diethylenetriaminepentaacetic acid (DTPA), N,N-bis(carboxymethyl)glycine (also called nitrile triacetic acid, NTA), l,2-bis(o-aminophenoxy)ethane-N, N,N',N'-tetraacetic acid (BAPTA), 2,3-dimer-capto-l-propanol (dimercaprol), porphin and heme. For example, EDTA forms a complex with most monovalent, divalent, trivalent and tetravalent metal ions, such as, for example, silver (Ag ⁺ ), calcium (Ca ²⁺ ), manganese (Mn ²⁺ ), copper (Cu ²⁺ ), iron (Fe ²⁺ ), cobalt (Co ⁺ ) and zirconium (Zr ⁴⁺ ), while BAPTA is Ca ²⁺ specific. As an illustrative example, the standard method used in the art is the formation of a complex between an oligohistidine label and copper (Cu ²⁺ ), nickel (Ni ²⁺ ), cobalt (Co ²⁺ ) or zinc (Zn ²⁺ ) ions, which presented by chelating nitrile triacetic acid (NTA).

In some embodiments, the binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent) contains a calmodulin binding peptide, and the reagent contains a multimeric calmodulin, as described in, for example, US Pat. No. 5,985,658. In some embodiments, The binding partner C that is contained in the agent (e.g., a receptor binding agent or selective agent) contains a FLAG peptide, and the reagent contains an antibody that binds to the FLAG peptide, e.g., a FLAG peptide that binds to a monoclonal antibody 4E11 as described in US Pat. . In some cases, disruption of all of these binding complexes can be accomplished by metal ion chelation, eg calcium chelation, eg by adding EDTA or EGTA. In some embodiments, calmodulin, antibodies such as 4E11, or chelated metal ions or free chelators can be multimerized using conventional methods, for example, by biotinylation and complexation with streptavidin or avidin or their oligomers, or by introducing carboxylic acids into the polysaccharide in the first step. residues, for example, dextran, essentially as described in Noguchi, et al. Bioconjugate Chemistry (1992) 3, 132-137, and second step coupling of calmodulin or antibodies or chelated metal ions or free chelators via primary amino groups with carboxyl groups in a polysaccharide, eg dextran, backbone using conventional carbodiimide chemistry. In some such embodiments, the binding between the binding partner C that is contained in the agent (eg, a receptor binding agent or selective agent) and one or more Z binding sites of the reagent can be broken by metal ion chelation. Metal chelation, for example, can be done by adding EGTA or EDTA.

In some embodiments, an agent (eg, a receptor binding agent or selective agent) that specifically binds to a cell surface molecule, for example, may be an antibody, fragment thereof, or protein binding molecule with antibody-like functions. In some embodiments, the B site of the agent is an antibody-combining site, such as being or containing one or more complementarity-determining regions (CDRs) of an antibody. Examples of (recombinant) antibody fragments include, but are not limited to, Fab fragments, Fv fragments, single chain Fv fragments (scFv), divalent antibody fragment such as (Fab)2' fragment, diabodies, tribodies (Iliades, P. et al, FEB S Lett (1997) 409, 437-441), decatela (Stone, E. et al, Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L.J. et al, Trends Biotechnol (2003), 21, 11, 484-490), single domain antibodies (nanobodies®). In some embodiments, the agent (eg, a receptor binding agent or selective agent) may contain a divalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein, which is also known as "duocalin".

In some embodiments, an agent (eg, a receptor binding agent or a selective agent) may have a single B binding site, ie. It may be monovalent. Examples of monovalent agents (eg, receptor binding agents or selective agents) include, but are not limited to, a monovalent antibody fragment, a protein binding molecule with antibody-like binding properties, or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to, a Fab fragment, an Fv fragment, single domain antibodies such as those derived from camelid nanobodies®, and a single chain Fv fragment (scFv), including a divalent single chain Fv fragment.

In some embodiments, the agent is an antibody or an antigen-binding fragment thereof, such as Fab fragments, Fv fragments, single chain Fv fragments (scFv), single domain antibodies, e.g. )2'-fragment. In some embodiments, the agent is, or is derived from, a parent antibody known to bind to a cell molecule of interest. Various anti-cell surface antibody molecules, or fragments thereof, are well known in the art, and any of a variety of such antibodies can be used as agents in the methods of this document. In some embodiments, the agent is an antibody, or fragment thereof, that contains one or more amino acid substitutions in the variable heavy chain of the parent or reference antibody, e.g., to generate an affinity-altered antibody, or that exhibits a reasonably fast dissociation rate as described above. For example, an example of such mutations is known in the context of anti-CD4 antibody 13B8.2 mutants (see, for example, US Pat. No. 7,482,000, US Patent Application No. US 2014/0295458 or International Patent Application No. WO 2013/124474), and any such mutations can be generated from another parent or reference antibody.

In some aspects, agents (e.g., a receptor binding agent or selective agent) that may be monovalent, for example, contain a monovalent antibody fragment or a monovalent artificial binding molecule (proteinaceous or otherwise), such as a lipocalin family polypeptide mutein (also known as Anticalin®), or a divalent molecule such as an antibody or fragment in which both binding sites are left, such as the F(ab') ₂ fragment.

An example of a protein binding molecule with antibody-like functions includes a lipocalin family polypeptide mutein (see, for example, WO 03/029462, Beste et al, Proc. Natl. Acad. Sci. U.S.A. (1999) 96, 1898-1903). Typically, lipocalins such as bilin-binding protein, human neutrophil gelatinase associated lipocalin, human Apo lipoprotein D, or human tear fluid lipocalin have natural ligand binding sites that can be modified to bind to a given target. Additional examples of a protein binding molecule with antibody-like binding properties that can be used as an agent (eg, receptor binding agent or selective agent) that specifically binds to a cell surface molecule include, but are not limited to, so-called glutels (see, for example, , international patent application WO 96/23879), proteins based on an ankyrin backbone (Mosavi, L.K. et al, Protein Science (2004) 13, 6, 1435-1448) or crystalline backbone (for example, international patent application WO 01 /04144), proteins described in Skerra, J. Mol. Recognize. (2000) 13, 167-187, Adnectins, tetranectins and avimers. Typically, avimers, including polyvalent avimer proteins, identified by exon shuffling of the human receptor family of domains, include so-called A-domains that occur as chains of multiple domains in several cell surface receptors (Silverman, J. et al, Nature Biotechnology (2005) 23, 1556-1561). Adnectins, usually derived from the human fibronectin domain, typically contain three loops that can be engineered for immunoglobulin-like binding to targets (Gill, D.S. & Damle, N.K. Current Opinion in Biotechnology (2006) 17, 653-658). Tetranectins, usually derived from the corresponding human homotrimeric protein, also typically contain loop regions in the C-type lectin domain that can be engineered for desired binding. Peptoids, which in some cases can act as protein ligands, are usually oligo(N-alkyl)glycines, which differ from peptides in that the side chain is linked to the amide nitrogen rather than to the carbon atom. Peptoids are generally resistant to proteases and other modifying enzymes and can have significantly higher cell permeability than peptides (see, for example, Kwon, Y.-U. and Kodadek, T. J. Am. Chem. Soc. (2007) 129, 1508- 1509).

Additional examples of suitable protein binding molecules include, but are not limited to, EGF-like domain, Kringle domain, fibronectin type I domain, fibronectin type II domain, fibronectin type III domain, PAN domain, Gla domain, SRCR domain, Kunitz/trypsin inhibitor domain bovine pancreas, tendamistat, Kazal type serine protease inhibitor domain, Trefoil (P-type) domain, von Willebrand factor C-type domain, anaphylatoxin-like domain, CUB domain, thyroglobulin type I repeat, LDL receptor A class domain, Sushi domain , Link domain, type I thrombospondin domain, immunoglobulin domain or immunoglobulin-like domain (eg, domain antibodies or camel heavy chain antibodies), C-type lectin domain, MAM domain, von Willebrand factor A-type domain, somatomedin B domain, WAP core domain -type with four disulfide bonds, C-type F5/8 domain, hemopexin domain, SH2 domain, SH3 domain, laminin-type EGF-like domain, C2 domain, "Kappabodies" (Ill et al. Protein Eng (19 97) 10, 949-57, so-called "mini-antibodies" (Martin et al, EMBO J (1994) 13, 5303-5309), diabody (Holliger et al, PNAS USA (1993)90, 6444-6448), so-called "Janusis" (Traunecker et al, EMBO J (1991) 10, 3655-3659 or Traunecker et al, Int J Cancer (1992) Suppl 7, 51-52), nanobody, microantibody, affilin, affibody, nottin, ubiquitin, protein zinc finger, autofluorescent protein, DARPins or leucine rich repeat protein. In some embodiments, the nucleic acid molecule with antibody-like functions may be an aptamer. Typically, an aptamer folds into a specific three-dimensional motif and shows high affinity for that target structure.

In some embodiments, one or more agents, for example, agents containing a binding partner, such as binding partner C, are attached, combined, and/or associated with the particulate oligomeric reagent. In specific embodiments, one or more agents are reversibly attached, combined and/or associated with the particulate oligomeric reagent. In some embodiments, one or more agents are attached, combined, and/or associated, eg, reversibly associated with the particulate oligomeric reagents by contacting, treating and/or incubating one or more agents with the particulate oligomeric reagents. In some embodiments, the processing, contact and/or incubation is carried out with stirring and/or rocking, for example, gentle rocking and/or stirring.

In some embodiments, one or more agents are incubated, treated, and/or contacted with particulate oligomeric reagents for, for about, or for at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, 120 minutes, 4 hours, 6 hours, 8 hours, 12 hours, 16 hours or 24 hours. In some embodiments, one or more agents are incubated, treated, and/or contacted with particulate oligomeric reagents for between 1 minute and 12 hours, between 1 minute and 1 hour, between 1 minute and 30 minutes, between 10 minutes and 60 minutes, between 10 minutes and 20 minutes, between 1 o'clock and 3 o'clock, between 1 o'clock and 2 o'clock, or between 6 o'clock and 12 o'clock. In some embodiments, one or more agents are incubated, treated, and/or contacted with particulate oligomeric reagents for 5 minutes to 30 minutes, or for or about 15 minutes.

In specific embodiments, one or more agents are incubated, processed, and/or contacted with particulate oligomeric reagents at a temperature of at least 4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C, 37°C, 39°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100°C. In some embodiments, one or more agents are incubated, processed and/or contacted with particulate oligomeric reagents at a temperature between 4°C and 39°C, between 10°C and 37°C, between 10°C and 25°C. C, between 20°C and 30°C, between 24°C and 39°C or between 40°C and 100°C. In specific embodiments, one or more agents are incubated, processed and/or contacted with particulate oligomeric reagents at room temperature. In some embodiments, one or more agents are incubated, processed, and/or contacted with particulate oligomeric reagents at or about 23°C, 24°C, 25°C, or 26°C ± 2°C, ± 1° C, ± 0.5°C, ± 0.2°C, ± 0.1°C, ± 0.05°C or ± 0.01°C.

In some embodiments, one or more agents are incubated, treated, and/or contacted with particulate oligomeric reagents in an amount of, about, or at least 0.1 µg, 0.2 µg, 0.3 µg, 0.4 mcg, 0.5 mcg, 0.6 mcg, 0.7 mcg, 0.8 mcg, 0.9 mcg, 1.0 mcg, 1.2 mcg, 1.4 mcg, 1.6 mcg, 1.8 µg, 2.0 µg, 2.2 µg, 2.4 µg, 2.6 µg, 2.8 µg, or 3.0 µg agents per 0.5 µg, 1.0 µg, 1.5 µg , 2.0 μg, 2.5 μg, 3.0 μg, 4.0 μg, 5.0 μg, 6 μg, 7 μg, 8 μg, 9 μg, or 10 μg particulate oligomeric reagent. In specific embodiments, one or more agents are incubated, processed, and/or contacted with particulate oligomeric reagents in an amount equal to or approximately equal to 1.0 μg of agents per 2 μg, 3 μg, 4 μg, or 5 μg of oligomeric reagent per the form of particles.

In some embodiments, one or more agents, e.g., agents containing a binding partner, such as binding partner C, are attached, combined, and/or associated with the particulate oligomeric reagent by incubation, processing, and/or contacting the oligomeric reagents. in the form of particles with one or more agents. In some embodiments, one or more agents are incubated, processed, and/or contacted with particulate oligomeric reagents in an amount equal to or approximately equal to 1.0 μg of agents per 2 μg, 3 μg, 4 μg, or 5 μg of oligomeric reagent per in the form of particles for 1 minute to 1 hour at a temperature between 4°C and 39°C, between 10°C and 25°C or between 20°C and 30°C. In specific embodiments, one or more agents are incubated, treated, and/or contacted with particulate oligomeric reagents in an amount equal to or approximately equal to 1.0 μg of agents per 3 μg of particulate oligomeric reagent for 5 minutes to 30 minutes at temperatures between 10°C and 25°C. In some embodiments, the implementation of one or more agents are agents described in this document, for example, in section II-C-3. In some embodiments, the agent is an anti-CD3 and/or anti-CD28 antibody or antigen-binding fragment thereof, eg an antibody or antigen-binding fragment thereof, which contains a binding partner, eg streptag. In specific embodiments, the one or more agents are an anti-CD3 and/or anti-CD28 Fab containing a binding partner, eg, a streptavidin binding peptide such as Strep-tagII.

a. Receptor binding agents

In some embodiments, the agent is a receptor binding agent. In some embodiments, the receptor-binding agent binds to a molecule (eg, a receptor) on the surface of a cell, and this binding between the agent and the molecule is capable of inducing or modulating a signal in the cells. In some cases, a cell surface molecule (eg, a receptor) is a signaling molecule. In some such instances, the receptor binding agent is capable of specifically binding to a signaling molecule expressed by one or more cells. In some instances, a receptor binding agent is a stimulatory agent, which can be any agent that is capable of inducing a signal in a cell (eg, T cell) when bound to a cell surface molecule, such as a receptor. In some embodiments, the signal may be immunostimulatory, in which case the receptor-binding agent or stimulatory agent is capable of inducing or modulating a signal that is involved in or that stimulates an immune response by a cell (e.g., T cell), e.g., increases proliferation or multiplication of immune cells, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other immune cell functional activities. In some embodiments, the signal may be inhibitory, in which case the receptor binding agent or stimulatory agent is capable of inducing or modulating a signal in a cell (e.g., T cell) that is involved in or that inhibits an immune response, e.g., inhibits or reduces proliferation or multiplication. immune cells, immune cell activation, immune cell differentiation, cytokine secretion, cytotoxic activity, or one or more other immune cell functional activities.

In some embodiments, the implementation of the receptor-binding agent, for example, the stimulating agent is the first receptor-binding agent, for example, the first stimulating agent. In some aspects, the first receptor-binding agent, for example, the first stimulatory agent, binds to a receptor molecule on the surface of cells. Thus, in some cases, the first receptor-binding agent, for example, the first stimulatory agent, causes or modulates a signal. In some aspects, the induction or modulation of the signal by the first receptor-binding agent, for example, the first stimulatory agent, affects the activation, stimulation and/or reproduction (proliferation) of cells. Thus, in some cases, the first receptor-binding agent, for example, the first stimulatory agent, provides the cells with a primary activation signal, thus activating the cells.

In some embodiments, the cell population may be a lymphocyte population, including but not limited to a B cell population, a T cell population, or a natural killer cell population. Illustrative examples of cell populations are B cells carrying CD40 or CD137 (both cell populations can proliferate by binding only the first agent that provides an activation signal, e.g. , Zhang et al., 2010, J Immunol, 184:787-795)). Other illustrative examples of agents (either the first or the second) that can be used to expand B cells are agents that bind to IgG, CD19, CD28, or CD14, such as αCD19, αIgG, αCD28, or αCD14 antibody molecules. It is also contemplated that the first or second B cell proliferating agents may contain ligands for toll-like receptors or interleukins such as IL-21 (see, for example, Dienz O, et al. 2009. J. Exp. Med. 206 :69). It should be noted that lipopolysaccharide-dependent activation of B cells is also included in the present invention, since lipopolysaccharide can also be used as the first agent, and within the scope of the present invention it can be provided with a C1 binding partner.

Other illustrative examples of suitable cell populations include a population of T cells that proliferate upon activation by binding a first agent to TCR/CD3 and binding a second agent to an accessory molecule on the T cell, such as CD28. In this case, the first agent stimulates the TCR/CD3 complex-associated signal in T cells, and the second agent provides a secondary stimulus by binding to CD28 as an accessory molecule. Agents that can be used to expand T cells can also include interleukins such as IL-2, IL-7, IL-15 or IL-21 (see, for example, Cornish et al. 2006, Blood. 108(2 ):600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22):12670-12674, Battalia et al, 2013, Immunology, 139(1):109-120). Other illustrative examples of agents that can be used to expand T cells are agents that bind to CD8, CD45, or CD90, such as αCD8, αCD45, or αCD90 antibodies. Illustrative examples of a population of T cells, including antigen-specific T cells, helper T cells, cytotoxic T cells, memory T cells (an illustrative example of memory T cells are CD62L ⁺ CD8+ specific central memory T cells) or regulatory T- cells (an illustrative example of Tregs are CD4 ⁺ CD25 ⁺ CD45RA+ Tregs).

Another illustrative example of a suitable cell population includes natural killer (NK) cells, which can, for example, be propagated with agents that bind to CD16 or CD56, such as, for example, αCD16 or αCD56 antibodies. An exemplary example of such an αCD16 antibody is the 3G8 antibody with the VH sequence set forth in SEQ ID NO: 52 and the VL sequence set forth in SEQ ID NO: 53 (see, e.g., Hoshino et al, Blood. 1991 Dec 15;78(12 ):3232-40.). Another agent that can be used to expand NK cells may be IL-15 (see, for example, Vitale et al. 2002. Anatomical Record. 266:87-92). Another illustrative example of a suitable cell population includes monocytes, which can, for example, be propagated using an agent that binds to CD14, such as an αCD14 antibody molecule.

In some aspects, the receptor-binding agent, for example, a stimulatory agent, specifically releases a molecule expressed on the surface of target cells, in which the molecule is a TCR or a chimeric antigen receptor. For example, the molecule expressed on the surface of the target cell is selected from a T-cell or B-cell antigen receptor complex, CD3 chain, CD3 zeta, an antigen-binding portion of a T-cell receptor or B-cell receptor, or a chimeric antigen receptor. In some instances, the receptor-binding agent releases class I peptide:MHC complexes. In some aspects, a receptor binding agent, eg, a stimulatory agent, specifically binds to an antibody portion of a recombinant receptor, eg, CAR. In some instances, the recombinant receptor antibody portion includes at least a portion of an immunoglobulin constant region, eg, a hinge region, eg, an IgG4 hinge region, and/or a CH1/CL and/or an Fc region. In some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some cases, the reagent is loaded with αIgG, which recognizes the IgG4 spacer.

In some embodiments, the implementation of the first receptor-binding agent, for example, the first stimulatory agent, can stimulate associated with the TCR/CD3 complex signal in cells, such as T-cells. In some aspects, the first receptor-binding agent, such as the first stimulatory agent, may be a binding agent that specifically binds CD3. In some instances, the first receptor-binding agent, e.g., the first stimulatory agent, that specifically binds CD3 may be selected from the group consisting of an anti-CD3 antibody, a bivalent anti-CD3 antibody fragment, a monovalent anti-CD3 antibody fragment, and a CD3 protein binding molecule with antibody-like binders. properties. The bivalent antibody fragment may be a (Fab) ₂ ' fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, a nanobody (sdAntibody) and a single chain Fv fragment (scFv ). In some instances, the CD3 protein binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, an ankyrin backbone protein, a crystalline backbone protein, adnectin, darpin, or avimer.

In some embodiments, an anti-CD3 Fab can be derived from a CD3-binding monoclonal antibody produced by the OKT3 hybridoma cell line (ATCC® CRL-8001™; see also US Pat. No. 4,361,549). The heavy chain variable domain and the light chain variable domain of the OKT3 anti-CD3 antibody are described in Arakawa et al J. Biochem. 120, 657-662 (1996) and contain the amino acid sequences shown in SEQ ID NOs: 31 and 32, respectively.

In some aspects, the receptor binding agent, eg a stimulant agent, is a second receptor binding agent, eg a second stimulatory agent. In some instances, a second receptor binding agent, eg, a second stimulatory agent, binds to a cell surface molecule, such as a cell surface molecule, eg, a receptor molecule. In some embodiments, the implementation of the second receptor-binding agent, for example, the second stimulatory agent, is able to enhance, attenuate or modify the signal delivered by the first molecule. In some embodiments, the implementation of the second receptor-binding agent, for example, the second stimulatory agent, causes or modulates the signal, for example, a second or additional signal. In some aspects, the second receptor binding agent, eg, the second stimulatory agent, can enhance or activate the signal induced by the first receptor binding agent, eg, the first stimulatory agent. In some embodiments, a second receptor binding agent, eg, a second stimulatory agent, binds to an accessory molecule and/or can stimulate or induce an additional or secondary signal in a cell. In some aspects, a second receptor binding agent, such as a second stimulatory agent, binds to a costimulatory molecule and/or provides a costimulatory signal.

In some embodiments, a receptor-binding agent, e.g., a stimulatory agent, which may be a second receptor-binding agent, e.g., a second stimulatory agent, binds, e.g., specifically binds, to a second molecule, which may be a costimulatory molecule, an accessory molecule, a cytokine a receptor, a chemokine receptor, an immune checkpoint molecule, or a member of the TNF family or the TNF receptor family.

In some embodiments, the molecule on the cell, such as a T cell, may be CD28, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD28. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) that specifically binds CD28, may be selected from the group consisting of an anti-CD28 antibody, a bivalent antibody fragment against CD28, a monovalent anti-CD28 antibody fragment, and a CD28 protein-binding molecule with antibody-like binding properties. The divalent antibody fragment may be a (Fab)2' fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). a CD28 protein binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, an ankyrin backbone protein, a crystalline backbone protein, adnectin, and avimer. In some instances, the receptor-binding agent, eg, the first, second, and/or additional receptor-binding agent, eg, stimulatory agent, that specifically binds the receptor is an antibody or an antigen-binding fragment thereof, eg, a Fab fragment.

In some embodiments, the receptor-binding agent, e.g., a stimulatory agent, can be a ligand that binds, e.g., specifically binds, to a receptor, e.g., a cell surface molecule that stimulates or activates T cell activation signal 2, e.g., a co-stimulatory signal , when binding the agent. In some embodiments, the receptor binding agent, such as the first, second, and/or additional receptor binding agent, is an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, variant, or modified form thereof. In some embodiments, the implementation of the receptor-binding agent, for example, a stimulatory agent, may be the extracellular domain of the endogenous ligand and/or cognate ligand or part thereof.

In some embodiments, a receptor binding agent, e.g., a stimulatory agent, is capable of binding to any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L and LIGHT. In some embodiments, the receptor-binding agent, such as a stimulatory agent, can be an antibody, a divalent antibody (e.g., F(ab') ₂ fragment or a divalent single chain Fv fragment), a monovalent antibody (e.g., Fab fragment, Fv- fragment or single chain Fv fragment (scFv)) or ligand for any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL , CD30L and LIGHT, and such an agent is able to induce or model a signal in cells upon binding a receptor-binding agent, for example, a stimulatory agent, to the molecule.

In some embodiments, the receptor binding agent, e.g., a stimulatory agent, may be a ligand for any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70 ), 4-1BBL, CD30L, and LIGHT, such an agent being able to induce or model a signal in cells upon binding a receptor-binding agent, such as a stimulatory agent, to the molecule. In some embodiments, the implementation of the receptor-binding agent, for example, a stimulatory agent, may be the extracellular domain of the endogenous ligand and/or cognate ligand or part thereof. For example, in some embodiments, the implementation of the receptor-binding agent is or contains an extracellular domain or part thereof, B7-1 (CD80), B7-2 (CD86), ICOS-L, PD-L1, OX40L, CD27, 4-1BB ( CD137) and/or CD30.

In some embodiments, a receptor-binding agent, such as a stimulatory agent, is capable of specifically binding to a molecule on the surface of target cells other than CD28, CD3, CD137, or CD40. In some instances, binding of a receptor-binding agent, such as a stimulatory agent, to a molecule on the surface of target cells other than CD28, CD3, CD137, or CD40 induces or modulates a signal in the target cells and/or alters target cell function, creating thus cultured target cells.

In some embodiments, a receptor-binding agent, e.g., a stimulatory agent, is capable of binding to any one or more members of the TNF family or TNF receptor family, e.g., a member of the TNF receptor superfamily, and/or a Wnt receptor or co-receptor, e.g., the Frizzled receptor family ( fz).

In some embodiments, the molecule on the cell is a member of the TNF receptor superfamily, such as tumor necrosis factor receptor 1 (CD120a), tumor necrosis factor receptor 2 (CD120b), lymphotoxin beta receptor (CD18), OX40 (CD134), CD40 (Bp50) , Fas receptor (Apo-1, CD95), decoy receptor 3 (TR6, M68), CD27 (S152, Tp55), CD30 (Ki-1), 4-1BB (CD137), death receptor 4 (TRAILR1, Apo -2, CD261), death receptor 5 (TRAILR2, CD262), decoy receptor 1 (TRAILR3, LIT, TRID, CD263), decoy receptor 2 (TRAILR4, TRUNDD, CD264), RANK (CD265), osteoprotegerin (OCIF, TR1), TWEAK receptor (Fn14, CD266), TACI (IGAD2, CD267), BAFF receptor (CD268), herpesvirus entry mediator (ATAR, TR2, CD270), nerve growth factor receptor (p75NTR, CD271), B-cell maturation antigen (TNFRSF13A, CD269), glucocorticoid-induced TNFR (AITR, CD357), TROY (TAJ, TRADE), death receptor 6 (CD358), death receptor 3 (Apo-3, TRAMP, LARD, WS-1), or ectodysplasin A2 receptor (XEDAR).

In some instances, the receptor-binding agent that specifically binds a TNF receptor superfamily protein is an antibody or an antigen-binding fragment thereof, such as a Fab fragment. In some embodiments, a receptor binding agent that specifically binds a TNF receptor superfamily protein can be a ligand that binds, eg, specifically binds, to the receptor and/or the extracellular domain or a portion thereof. In some embodiments, the ligand is or contains TNFα, lymphotoxin beta (TNF-C), OX40L, CD154, FasL, FasL, LIGHT, TL1A, CD70, Siva, CD153, ligand 4-1BB, TRAIL, RANKL, TWEAK, APRIL, BAFF , CAMLG, BAFF, LIGHT, NGF, BDNF, NT-3, NT-4, BAFF, GITR ligand, TL1A or EDA-A2, or the extracellular domain or part thereof of any of the transmembrane proteins.

In some embodiments, the molecule on the cell is a Wnt receptor or co-receptor, receptors like the Frizzled (Fz) family receptor, lipoprotein (LRP)-5/6 receptor-related protein, receptor tyrosine kinase (RTK), and receptor-coupled orphan receptor 2 (ROR2). In some embodiments, the molecule on the cell is, for example, Frizzled-1 (FZD1), Frizzled-2 (FZD2), Frizzled-3 (FZD3), Frizzled-4 (FZD4), Frizzled-5 (FZD5), Frizzled-6 (FZD6), Frizzled-7 (FZD7), Frizzled-8 (FZD8), Frizzled-9 (FZD9) or Frizzled-10 (FZD10).

In some instances, the receptor-binding agent that specifically binds a Wnt receptor or co-receptor is an antibody or an antigen-binding fragment thereof, such as a Fab fragment. In some embodiments, a receptor binding agent that specifically binds a Wnt receptor or co-receptor may be a ligand that binds, eg, specifically binds, to the receptor. In some embodiments, the ligand is or contains, for example, WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, or WNT16.

In some embodiments, a receptor binding agent, e.g., a stimulatory agent, is capable of binding to any one or more of CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 and HVEM. In some embodiments, the receptor-binding agent, such as a stimulatory agent, can be an antibody, a divalent antibody (e.g., F(ab') ₂ fragment or a divalent single chain Fv fragment), a monovalent antibody (e.g., Fab fragment, Fv- fragment or single chain Fv fragment (scFv)) or ligand for any one or more of CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT , CD27, OX40, and HVEM, wherein such an agent is capable of inducing or modeling a signal in cells upon binding a receptor-binding agent, such as a stimulatory agent, to the molecule.

In some embodiments, the receptor binding agent, e.g., a stimulatory agent, can be a ligand for any one or more of CD28, 4-1BB (CD137), CD40, CD40L, T cell activation linker (LAT), CD27, OX40 ( CD134) and a herpesvirus entry mediator (HVEM), which agent is capable of inducing or modeling a signal in cells upon binding a receptor-binding agent, such as a stimulatory agent, to the molecule. In some embodiments, the implementation of the receptor-binding agent, for example, a stimulatory agent, may be the extracellular domain of the endogenous ligand and/or cognate ligand or part thereof. For example, in some embodiments, the receptor binding agent is or contains an extracellular domain or a portion thereof, B7-1 (CD80), B7-2 (CD86), 4-1BBL, CD40, CD40L, CD27L (CD70), and/or OX40L .

In some embodiments, the molecule on the cell, such as a T cell, may be CD28, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD28.

In some embodiments, the molecule on the cell, such as a T cell, may be CD28, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD28. In some aspects, a receptor-binding agent, such as a stimulatory agent (eg, which may be a second receptor-binding agent, such as a second stimulatory agent) that specifically binds CD28, may be selected from the group consisting of an anti-CD28 antibody, a bivalent antibody fragment against CD28, a monovalent anti-CD28 antibody fragment, and a CD28 protein-binding molecule with antibody-like binding properties. The divalent antibody fragment can be a F(ab') ₂ fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, a nanobody, and a single chain Fv fragment (scFv).

In some embodiments, the anti-CD28 Fab can be derived from the CD28.3 antibody (placed as a synthetic single chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al, BLOOD, 15 July 2003, Vol. 102, No. 2, pages 564-570), the heavy and light chains of which comprise SEQ ID NOs: 33 and 34, respectively.

In some embodiments, the molecule on the cell, such as a T cell, may be CD90, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD90. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) that specifically binds CD90, may be selected from the group consisting of an anti-CD90 antibody, a bivalent antibody fragment anti-CD90, a monovalent anti-CD90 antibody fragment, and a CD90 protein-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field. See, for example, anti-CD90 G7 antibody (Biolegend, cat. no. 105201).

In some embodiments, the molecule on the cell, such as a T cell, may be CD95, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD95. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) that specifically binds CD95, may be selected from the group consisting of an anti-CD95 antibody, a bivalent antibody fragment against CD95, a monovalent anti-CD95 antibody fragment, and a CD95 protein-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field. For example, in some aspects, the anti-CD90 antibody may be a monoclonal mouse anti-human CD95 CH11 (Upstate Biotechnology, Lake Placid, NY) or may be an anti-CD95 or anti-APO-1 mAb 7C11, for example, as described in Paulsen et al. Cell Death & Differentiation 18.4 (2011): 619-631.

In some embodiments, the molecule on the cell, such as a T cell or B cell, may be CD137 and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) specifically binds CD137. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) that specifically binds CD137, may be selected from the group consisting of an anti-CD137 antibody, a bivalent antibody fragment against CD137, a monovalent anti-CD137 antibody fragment, and a CD137 protein binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field. For example, the anti-CD137 antibody can be LOB12, IgG2a or LOB12.3, IgG1, as described in Taraban et al. Eur J Immunol. 2002 Dec;32(12):3617-27. See also, for example, US6569997, US6303121, Mittler et al. Immunol Res. 2004;29(1-3):197-208.

In some embodiments, the molecule on the cell, such as a B cell, may be CD40, and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), specifically binds CD40. In some aspects, the receptor binding agent (which may be a second receptor binding agent, e.g., a second stimulatory agent) that specifically binds CD40 may be selected from the group consisting of an anti-CD40 antibody, a bivalent anti-CD40 antibody fragment, a monovalent anti-CD40 antibody fragment, CD40 and CD40 protein binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field. For example, the anti-CD40 antibody can be Teneliximab and anti-human CD40 chimeric monoclonal antibody (Affymetrix cat. no. 14-0409-80) or any of those described, for example, in US 2002/0142358, US 2007/0077242, WO 2001/083755 , Zhang et al. 2010, J Immunol, 184:787-795.

In some embodiments, the molecule on the cell, such as a T cell, may be CD40L (CD154), and the receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) is specifically binds CD40L. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent) that specifically binds CD40L, may be selected from the group consisting of an anti-CD40L antibody, a bivalent antibody fragment against CD40L, a monovalent anti-CD40L antibody fragment, and a CD40L protein-binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field. For example, in some aspects, the anti-CD40L antibody may be Hu5C8, as described in Blair et al. JEM vol. 191 No. 4 651-660. See also, for example, WO1999061065, US20010026932, US7547438, WO2001056603. In some embodiments, the molecule on the cell, e.g., a T cell, can be an inducible T cell costimulator (ICOS), T cell activation linker (LAT), CD27, OX40 (CD134), herpesvirus entry mediator (HVEM), CD90 and/or CD95, and a receptor binding agent, e.g., a stimulatory agent (eg, which may be a second receptor binding agent, e.g., a second stimulatory agent) specifically binds ICOS, LAT, CD27, CD134, HVEM, CD90 and/or CD95 , respectively. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be a second receptor binding agent, such as a second stimulatory agent), that specifically binds ICOS, LAT, CD27, CD134, HVEM, CD90, and/or CD95, may be selected from the group consisting of an antibody, a divalent antibody fragment thereof, a monovalent antibody fragment thereof, and a protein binding molecule with antibody-like binding properties. The antibody or antigen-binding fragment can be obtained from any known in this field.

In some embodiments, a molecule on a cell, such as a T cell, on which a receptor binding agent, which may be a second or additional receptor binding agent, binds to a cell surface molecule that stimulates or activates a cytokine signal, a chemokine signal, an adhesion signal cells, a T cell activation signal, or additional signals, such as environmental signals, when the agent binds. In some embodiments, the molecule on the cell, such as a T cell, to which the receptor binding agent specifically binds, which may be a second or additional receptor binding agent, is a cytokine receptor or a chemokine receptor. In some cases, the receptor binding agent, e.g., an additional receptor binding agent, is or contains an adhesion molecule, is a factor that induces cytokine production, chemokine production, expression of the adhesion molecule, and/or is involved in stimulating and/or modulating ancillary signal and/or an additional signal, such as an environmental signal.

In any of the embodiments provided herein, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., a stimulatory agent, may be a binding agent that specifically binds a receptor, e.g., a receptor expressed on the surface of the cell.

In some embodiments, a receptor-binding agent, e.g., a stimulatory agent, is an antibody or antigen-binding fragment thereof, e.g., an antibody or antigen-binding fragment thereof, that binds to a cell surface molecule that stimulates or activates a cytokine signal, chemokine signal, signal cell adhesion, a T cell activation signal, or additional signals, such as environmental signals, when the agent binds. In some instances, the receptor binding agent, such as a first, second, and/or additional receptor binding agent, such as a stimulatory agent that specifically binds the receptor, may be selected from the group consisting of an anti-receptor antibody, a bivalent anti-receptor antibody fragment, a a monovalent antibody against the receptor; and a protein receptor-binding molecule with antibody-like binding properties. The divalent antibody fragment can be a F(ab') ₂ fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). In some instances, the proteinaceous receptor-binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, an ankyrin backbone protein, a crystalline backbone protein, adnectin, darpin, or avimer. In some instances, the receptor-binding agent, eg, the first, second, and/or additional receptor-binding agent, eg, stimulatory agent, that specifically binds the receptor is an antibody or an antigen-binding fragment thereof, eg, a Fab fragment.

In some embodiments, the receptor-binding agent, e.g., a stimulatory agent, can be a ligand that binds, e.g., specifically binds, to a receptor, e.g., a cell surface molecule that stimulates or activates a cytokine signal, a chemokine signal, a cell adhesion signal, a activation of T cells or additional signals, such as environmental signals, when the agent binds. In some embodiments, the receptor binding agent, such as the first, second, and/or additional receptor binding agent, is an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, variant, or modified form thereof. In some embodiments, the implementation of the receptor-binding agent, for example, a stimulatory agent, may be the extracellular domain of the endogenous ligand and/or cognate ligand or part thereof.

In some embodiments, the implementation of the receptor-binding agent, for example, the second or additional receptor-binding agent, is or contains a ligand that specifically binds to a cytokine receptor. In some embodiments, the implementation of the receptor-binding agent, for example, the second or additional receptor-binding agent, is or contains a ligand of a cytokine receptor, for example, a cytokine or part thereof. Exemplary cytokine receptors include, but are not limited to, IL-2R, IL-7R, IL-21R, CD132 (common IL receptor chain gamma), IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR type I, IL-12R, IL-17R, TNFR1 and TNFR2. Illustrative ligands such as cytokines include, but are not limited to, IL-2, IL-7, IL-21, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, interferons type I (eg, IFNα and/or IFNβ), IL-12, IL-17, IL-9 and TNF, and their biologically active fragments.

In some embodiments, the implementation of the receptor-binding agent, for example, the second or additional receptor-binding agent, is an antibody or antigennegative fragment that specifically binds to a cytokine receptor. In some instances, the receptor binding agent, e.g., a second or additional receptor binding agent that specifically binds a cytokine receptor, may be selected from the group consisting of an anti-cytokine receptor antibody, a bivalent anti-cytokine receptor antibody fragment, a monovalent anti-cytokine receptor antibody fragment, and a protein a cytokine receptor-binding molecule with antibody-like binding properties. The divalent antibody fragment may be a F(ab') ₂ fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv).

In some embodiments, the implementation of the receptor-binding agent, for example, additional receptor-binding agent, is or contains a ligand that specifically binds to a chemokine receptor. In some embodiments, the receptor binding agent, eg, the second or additional receptor binding agent, is or contains a chemokine receptor ligand, eg, a chemokine, or a portion thereof. Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3, and CXCR4. Illustrative ligands, such as chemokines, include, but are not limited to, CXCL9, CXCL10, CCL19, CCL21, and CCL25, or biologically active fragments thereof.

In some embodiments, the implementation of the receptor-binding agent, for example, the second or additional receptor-binding agent, is an antibody or antigennegative fragment that specifically binds to a chemokine receptor. In some instances, the receptor binding agent, e.g., a second or additional receptor binding agent that specifically binds a chemokine receptor, may be selected from the group consisting of an anti-chemokine receptor antibody, a bivalent anti-chemokine receptor antibody fragment, a monovalent anti-chemokine receptor antibody fragment, and a protein-binding chemokine receptor molecule with antibody-like binding properties. The divalent antibody fragment may be a F(ab') ₂ fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv).

In some instances, the receptor-binding agent that specifically binds an adhesion molecule or factors that induce a cytokine is an antibody or an antigen-binding fragment thereof, such as a Fab fragment. In some embodiments, the receptor binding agent that specifically binds an adhesion molecule or factors that induce a cytokine may be a ligand that binds, eg, specifically binds, to the receptor. In some embodiments, the receptor binding agent is an endogenous ligand, a cognate ligand, a synthetic ligand, and/or a portion, variant, or modified form, an adhesion molecule, and/or a receptor. In some embodiments, the implementation of the receptor-binding agent, such as a stimulatory agent, may be the extracellular domain of the endogenous ligand and/or cognate ligand, or part thereof, which is itself a cell surface or transmembrane protein.

In some instances, the molecule on the cell, e.g., the adhesion molecule, is CD44, CD31, CD18/CD11a (LFA-1; full-length alpha and beta chain sequence given in SEQ ID NOs: 36 and 37, respectively), CD29, CD54 (ICAM-1), CD62L (L-selectin; full length sequence given in SEQ ID NO:38), CD29/CD49d (VLA-4; full length sequence given in SEQ ID NO:40), CD106 (VCAM-1; full-length sequence shown in SEQ ID NO:39), or is their biologically active fragment. In some embodiments, the receptor-binding agent is an antibody or antigen-binding fragment thereof that binds, e.g., specifically binds to an adhesion molecule, e.g., CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1) , CD62L (L-selectin), CD29/CD49d (VLA-4), CD106 (VCAM-1), or is their biologically active fragment. In some embodiments, a receptor binding agent is a ligand or portion thereof that binds, e.g., specifically binds to an adhesion molecule, e.g., CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), CD29/CD49d (VLA-4), CD106 (VCAM-1). Such receptor binding agents include an agent that is or contains the extracellular domain (ECD) of an endogenous ligand and/or a cognate ligand of an adhesion molecule, or a portion thereof. In some embodiments, the implementation of the receptor-binding agent is an extracellular domain of the adhesion molecule or part thereof, and can bind one or more adhesion molecules on the cell surface. Examples of such receptor binding agents include the extracellular domain of LFA-1α (ECD shown in SEQ ID NO: 54); LFA-1β (ECD given in SEQ ID NO: 55); L-selectin (ECD given in SEQ ID NO: 57); VCAM-1 (ECD given in SEQ ID NO: 56); and VLA-4 (ECD given in SEQ ID NO: 58) or any part thereof.

In some embodiments, the factor that causes cytokine production, chemokine production, and/or adhesion molecule expression is or contains a nuclear factor, such as orphan receptor gamma coupled to retinoic acid receptors (RORgamma) or RORalpha.

In some embodiments, the implementation of the receptor-binding agent, for example, the second or additional receptor-binding agent, is or contains a ligand that specifically binds to a cytokine receptor.

In some embodiments, the molecule on the cell, e.g., a T cell, can be IL-2R, IL-7R (CD127), IL-21R (CD360), common IL receptor gamma chain (γc; or CD132), IL-1R ( CD121), such as IL-1R1 or IL-1R2, IL-15R (CD215), interferon receptor gamma (IFNγR; CD119), tumor necrosis factor receptor alpha (TNFαR), including TNFR1 (CD120a) and TNFR2 (CD120b), IL -4R, IL-10R, type I interferon receptor, e.g., IFNα receptor (IFNAR), including IFNAR1 and IFNAR2, IL-17R (CD217) and/or IL-12R, and a receptor-binding agent, e.g., a stimulatory agent, specifically binds to the molecule. In some aspects, a receptor-binding agent, e.g., a stimulatory agent (e.g., which may be an additional receptor-binding agent, e.g., an additional stimulatory agent) that specifically binds a molecule to a cell, can be selected from the group consisting of an antibody, a divalent antibody fragment , a monovalent antibody fragment, and a protein binding molecule with antibody-like binding properties.

In some embodiments, a receptor binding agent, e.g., a stimulatory agent (e.g., which may be an additional receptor binding agent, e.g., an additional stimulatory agent), may contain a ligand of a stimulatory receptor or other receptor capable of inducing a signal in a cell, such as a ligand IL-2, IL-7 ligand, IL-21 ligand, IL-1 ligand, IL-15 ligand, IL-9 ligand, IFNγ ligand, TNFα ligand, IL-4 ligand, IL-10 ligand, IL-12 ligand, IL-17 ligand or biologically active portion or variant thereof. In some embodiments, the biologically active moiety or variant retains receptor binding activity and/or functional activity to modulate one or more signals to the receptor.

In some embodiments, a receptor binding agent, such as a stimulatory agent (eg, which may be an additional receptor binding agent, such as an additional stimulatory agent), may contain a stimulatory receptor or other receptor ligand capable of inducing a signal in the cell, such as an interferon type I, for example, IFNα, IFNβ, IFNκ, IFNδ, IFNε, IFNτ, IFNω and IFNζ (also known as limitin) or its biologically active part.

In some embodiments, the molecule on the cell, such as a T cell, may be CXCR3, CCR7, CXCR1, or CXCR4, and the receptor binding agent, e.g., a stimulatory agent (e.g., which may be an additional receptor binding agent, e.g., an additional stimulatory agent), binds specifically to CXCR3, CCR7, CXCR1, or CXCR4. In some aspects, a receptor binding agent, such as a stimulatory agent (eg, which may be an additional receptor binding agent, such as an additional stimulatory agent) that specifically binds to CXCR3, CCR7, CXCR1, or CXCR4, may be selected from the group consisting of an antibody , a bivalent antibody fragment, a monovalent antibody fragment, and a protein binding molecule with antibody-like binding properties.

In some embodiments, a receptor binding agent, e.g., a stimulatory agent (e.g., which may be an additional receptor binding agent, e.g., an additional stimulatory agent), may contain a ligand of a stimulatory receptor or other receptor capable of inducing a signal in a cell, such as a ligand CXCL9, CXCL10 ligand, CCL19 ligand, CCL21 ligand, CCL25 ligand, or a biologically active portion thereof that retains receptor binding activity and/or functional activity to modulate one or more signals to the receptor.

In some instances, the receptor binding agent, e.g., an additional receptor binding agent, is or contains an adhesion molecule, or is a factor that induces cytokine production, chemokine production, adhesion molecule expression, and/or is involved in stimulating and/or modulating ancillary signal and/or additional signal. In some embodiments, the molecule on the cell, such as a T cell, may be CD62L, and the receptor binding agent, such as a stimulatory agent (eg, which may be an additional receptor binding agent, such as an additional stimulatory agent), specifically binds CD62L . In some embodiments, the molecule on the cell, such as a T cell, may be RORγt, and the receptor binding agent, such as a stimulatory agent (eg, which may be an additional receptor binding agent, such as an additional stimulatory agent), specifically binds RORγt . In some embodiments, the molecule on the cell, such as a T cell, may be RORα, and the receptor binding agent, such as a stimulatory agent (eg, which may be an additional receptor binding agent, such as an additional stimulatory agent), specifically binds RORα .

In some embodiments, a receptor binding agent, e.g., a stimulatory agent, is capable of binding to any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L, and LIGHT, and the receptor binding agent, e.g., a stimulatory agent, can be an antibody, a divalent antibody (e.g. (Fab)2' fragment or a divalent single chain Fv fragment), a monovalent antibody (e.g. a Fab fragment , Fv fragment or single chain Fv fragment (scFv)) or a ligand for any one or more of CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4, ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L and LIGHT, and such an agent is able to induce or model a signal in cells upon binding a receptor-binding agent, for example, a stimulatory agent, to the molecule.

In some embodiments, a receptor-binding agent, such as a stimulatory agent, is capable of specifically binding to a molecule on the surface of target cells other than CD28, CD3, CD137, or CD40. In some instances, binding of a receptor-binding agent, such as a stimulatory agent, to a molecule on the surface of target cells other than CD28, CD3, CD137, or CD40 induces or modulates a signal in the target cells and/or alters target cell function, creating thus cultured target cells.

In any of the above examples, the bivalent antibody fragment can be a (Fab) ₂ ' fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment ( scFv). In any of the above examples, the protein binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, an ankyrin backbone protein, a crystalline backbone protein, adnectin, nanobody, darpin, and avimer.

b. Selective agents

In some embodiments, the agent is a selective agent. In some embodiments, the selective agent binds to a cell surface molecule, such as a cell surface molecule. In some cases, the cell surface molecule is a selectable marker. In some such cases, the selective agent is able to specifically bind to a selectable marker expressed by one or more cells. In some embodiments, a selective agent or agents that are reversibly associated with a reagent can be used to facilitate cell selection or isolation.

In any of the embodiments provided herein, the receptor binding agent, e.g., the first, second, and/or additional receptor binding agent, e.g., a stimulatory agent, may be a binding agent that specifically binds a receptor, e.g., a receptor expressed on the surface of the cell. In some instances, the receptor binding agent, e.g., a first, second, and/or additional receptor binding agent, e.g., a stimulatory agent, that specifically binds the receptor may be selected from the group consisting of an anti-receptor antibody, a bivalent anti-receptor antibody fragment, a monovalent antibodies against the receptor and a protein receptor-binding molecule with antibody-like binding properties. The divalent antibody fragment can be a F(ab') ₂ fragment or a divalent single chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv). In some instances, the proteinaceous receptor-binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, ankyrin backbone protein, crystalline backbone protein, adnectin, nanobody, darpin, or avimer. In some instances, the receptor-binding agent, eg, the first, second, and/or additional receptor-binding agent, eg, stimulatory agent, that specifically binds the receptor is an antibody or an antigen-binding fragment thereof, eg, a Fab fragment.

In some aspects, a cell surface molecule, such as a selectable marker, may be an antigen that defines a desired population or subset of cells, such as a population or subset of blood cells, such as lymphocytes (eg, T cells, T helpers, such as CD4+ T- helper cells, B cells, or natural killer cells), monocytes, or stem cells, such as CD34-positive peripheral stem cells or Nanog or Oct-4 expressing stem cells. In some embodiments, the selectable marker may be a marker expressed on the surface of T cells or a subset of T cells, such as CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO. Examples of T cells include cells such as CMV-specific CD8+ T lymphocytes, cytotoxic T cells, memory T cells, and regulatory T cells (Treg). Illustrative Tregs include CD4 CD25 CD45RA Tregs, and illustrative memory T cells include CD62L CD8+ specific central memory T cells. The cell surface molecule, for example a selectable marker, may also be a tumor cell marker.

In some embodiments, the selection marker may be CD4, and the selection agent specifically binds CD4. In some aspects, the selective agent that specifically binds CD4 can be selected from the group consisting of an anti-CD4 antibody, a bivalent anti-CD4 antibody fragment, a monovalent anti-CD4 antibody fragment, and a CD4 protein-binding molecule with antibody-like binding properties (e.g., anticalin or nanobody). In some embodiments, an anti-CD4 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., a CD4 Fab fragment), can be derived from the 13B8.2 antibody or a functional 13B8.2 mutant that retains specific binding for CD4. For example, exemplary 13B8.2 or m13B8.2 antibody mutants are described in US Patent No. 7,482,000, US Patent Application No. US2014/0295458, or International Patent Application No. WO2013/124474; and Bes, C, et al. J Biol Chem 278, 14265-14273 (2003). The mutant Fab fragment, referred to as "ml3B8.2", carries the variable domain of the mouse CD4-binding antibody 13B8.2 and a constant domain containing a heavy chain human CH1 gamma constant domain and a kappa human light chain constant domain, as described in US Pat. 7,482,000. In some embodiments, an anti-CD4 antibody, e.g., a mutant of the 13B8.2 antibody, comprises an amino acid substitution H91A in the variable light chain, an amino acid substitution Y92A in the variable light chain, an amino acid substitution H35A in the variable heavy chain, and/or an amino acid substitution R53A in the variable heavy chain. heavy chain, each according to Kabat numbering. In some aspects, compared to the variable domains of Fab fragment 13B8.2 in ml3B8.2, the His residue at position 91 of the light chain (position 93 of SEQ ID NO: 30) is mutated to an Ala residue, and Arg at position 53 of the heavy chain (position 55 in SEQ ID NO: 29) is mutated in Ala. In some embodiments, a reagent that reversibly binds to an anti-CD4 antibody or fragment thereof is commercially available or is prepared from a commercially available reagent (e.g., catalog no. 6-8000-206 or 6-8000-205 or 6-8002- 100; IBA GmbH, Gottingen, Germany).

In some embodiments, the selection marker may be CD8, and the selection agent specifically binds CD8. In some aspects, the selective agent that specifically binds CD8 can be selected from the group consisting of an anti-CD8 antibody, a bivalent anti-CD8 antibody fragment, a monovalent anti-CD8 antibody fragment, and a CD8-binding protein molecule with antibody-like binding properties. In some embodiments, an anti-CD8 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., CD8 Fab fragment), can be derived from an OKT8 antibody (e.g., ATCC CRL-8014) or a functional mutant thereof that retains specific binding for CD8 . In some embodiments, a reagent that reversibly binds to an anti-CD8 antibody or fragment thereof is commercially available or derived from a commercially available reagent (e.g., Cat. No. 6-8003 or 6-8000-201; IBA GmbH, Gottingen, Germany ).

In some embodiments, the selection marker may be CD3, and the selection agent specifically binds CD3. In some aspects, the selective agent that specifically binds CD3 can be selected from the group consisting of an anti-CD3 antibody, a bivalent anti-CD3 antibody fragment, a monovalent anti-CD3 antibody fragment, and a CD3-binding protein molecule with antibody-like binding properties. In some embodiments, an anti-CD3 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., CD3 Fab fragment), can be derived from an OKT3 antibody (e.g., ATCC CRL-8001; see, e.g., Stemberger et al. PLoS One 2012; 7(4): e35798) or a functionally active mutant thereof which retains specific binding for CD3. In some embodiments, a reagent that reversibly binds to an anti-CD3 antibody or fragment thereof is commercially available, or derived from a commercially available reagent (e.g., Cat. No. 6-8000-201, 6-8001-100; IBA GmbH, Gottingen , Germany).

In some embodiments, the selection marker may be CD25 and the selection agent specifically binds CD25. In some aspects, the selective agent that specifically binds CD25 can be selected from the group consisting of an anti-CD25 antibody, a bivalent anti-CD25 antibody fragment, a monovalent anti-CD25 antibody fragment, and a CD25 protein-binding molecule with antibody-like binding properties. In some embodiments, an anti-CD25 antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., CD25 Fab fragment), can be derived from an FRT5 antibody (see, e.g., Stemberger et al. 2012) or a functional mutant thereof that retains specific binding for CD25. In some embodiments, a reagent that reversibly binds to an anti-CD4 antibody or fragment thereof is commercially available or is prepared from a commercially available reagent (e.g., catalog no. 6-8000-205 or 6-8000-207 or 6-8004- 050; IBA GmbH, Gottingen, Germany).

In some embodiments, the selection marker may be CD62L and the selection agent specifically binds CD62L. In some aspects, the selective agent that specifically binds CD62L can be selected from the group consisting of an anti-CD62L antibody, a bivalent anti-CD62L antibody fragment, a monovalent anti-CD62L antibody fragment, and a CD62L protein binding molecule with antibody-like binding properties. In some embodiments, an anti-CD62L antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., CD62L Fab fragment), can be derived from a DREG56 antibody (e.g., ATCC HB300; see, e.g., Stemberger et al. 2012) or functionally an active mutant that retains the specific binding for CD62L. In some embodiments, a reagent that reversibly binds to an anti-CD62L antibody or fragment thereof is commercially available, or is derived from a commercially available reagent (e.g., Cat. No. 6-8000-204 or 6-8005-050; IBA GmbH, Gottingen , Germany).

In some embodiments, the selection marker may be CD45RA and the selection agent specifically binds CD45RA. In some aspects, a selective agent that specifically binds CD45RA can be selected from the group consisting of an anti-CD45RA antibody, a bivalent anti-CD45RA antibody fragment, a monovalent anti-CD45RA antibody fragment, and a CD45RA protein-binding molecule with antibody-like binding properties. In some embodiments, an anti-CD45RA antibody, e.g., a bivalent antibody fragment or a monovalent antibody fragment (e.g., CD45RA Fab fragment), can be derived from a MEM56 antibody (e.g., Millipore 05-1413; see, e.g., Stemberger et al. 2012) or its functionally active mutant that retains the specific binding for CD45RA. In some embodiments, a reagent that reversibly binds to an anti-CD45RA antibody or fragment thereof is commercially available or obtained from a commercially available reagent (e.g., Cat. No. 6-8000-208 or 6-8007-050; IBA GmbH, Gottingen , Germany).

In some embodiments, the selection marker may be CD45RO and the selection agent specifically binds CD45RO. In some aspects, the selective agent that specifically binds to CD45RO can be selected from the group consisting of an anti-CD45RO antibody, a bivalent anti-CD45RO antibody fragment, a monovalent anti-CD45RO antibody fragment, and a CD45RO binding protein molecule with antibody-like binding properties. In some embodiments, a reagent that reversibly binds to an anti-CD45RO antibody or fragment thereof is commercially available, or derived from a commercially available reagent (e.g., Cat. No. 6-8000-209 or 6-8012-020; IBA GmbH, Gottingen , Germany).

In some embodiments, the selection marker may be CD154 and the selection agent specifically binds CD154. In some aspects, the selective agent that specifically binds CD154 can be selected from the group consisting of an anti-CD154 antibody, a bivalent anti-CD154 antibody fragment, a monovalent anti-CD154 antibody fragment, and a CD154 protein-binding molecule with antibody-like binding properties. In some embodiments, a reagent that reversibly binds to an anti-CD154 antibody or fragment thereof is commercially available, or is derived from a commercially available reagent (e.g., Cat. No. 6-8000-202 or 6-5510-050; IBA GmbH, Gottingen , Germany).

In some embodiments, the selectable marker may be CD16 and the selective agent specifically binds CD16. In some aspects, the selective agent that specifically binds CD16 can be selected from the group consisting of an anti-CD16 antibody, a bivalent anti-CD16 antibody fragment, a monovalent anti-CD16 antibody fragment, and a CD16 protein-binding molecule with antibody-like binding properties. In some embodiments, a reagent that reversibly binds to an anti-CD16 antibody or fragment thereof is commercially available or is derived from a commercially available reagent. In some aspects, the CD16 binding molecule comprises the heavy chain and/or light chain sequences set forth in SEQ ID NOs: 52 and 53, respectively.

In any of the above examples, the bivalent antibody fragment can be a (Fab) ₂ ' fragment or a bivalent single chain Fv fragment, while the monovalent antibody fragment can be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment ( scFv). In any of the above examples, the protein binding molecule with antibody-like binding properties can be an aptamer, a lipocalin family polypeptide mutein, glutelo, an ankyrin backbone protein, a crystalline backbone protein, adnectin, nanobody, darpin, and avimer.

III. Cell Culture Methods

Provided herein are cell culture methods that include incubating a composition containing target cells (e.g., T cells) in the presence of an agent (e.g., first or second receptor binding agents, e.g., stimulatory agents or selective agents) that is capable of bind to a molecule on the surface of target cells (eg, T cells) in the composition and which is reversibly associated with a reagent containing a plurality of binding sites capable of reversibly binding to the agent. In some embodiments, the reagent is a particulate oligomeric reagent, such as any of those described. In some embodiments, the implementation of the incubation is carried out under conditions in which the agent binds, for example, specifically binds, to a molecule on the cell. In some instances, for certain receptor binding agents (eg, stimulatory agents), such binding may induce or modulate a signal in target cells (eg, T cells) in the compositions, eg, a primary signal or an additional signal, as described. In some embodiments, binding of an agent to a molecule results in one or more of stimulation, activation, expansion (proliferation), and/or differentiation of target cells in the composition.

In some embodiments, the present method can be used to selectively induce ex vivo expansion of a population of cells, such as B cells, T cells, or natural killer cells. In some cases, stimulation can be performed in the absence of exogenous growth factors such as lymphokines and additional cells. In some embodiments, these cells, such as B cells or T cells, can be induced to proliferate without the need for antigen, thus providing a proliferating cell population, such as a T cell population, that is polyclonal with respect to antigen reactivity. The methods disclosed herein may involve slow proliferation of a selected population of T cells, such as CD4+ or CD8+ T cells, over an extended period of time to obtain a multiple of these cells relative to the original T cell population. In general, in the case of (clonal) propagation of a population of lymphocytes as described herein, all progeny may have the same antigenic specificity as the population of cells that is selected for propagation.

In some embodiments, the methods relate to expanding a population of antigen-specific T cells. In some embodiments, to obtain a population of antigen-specific T cells, the T cells are contacted with an antigen in a form suitable for triggering a primary activation signal in the T cell, i.e. The antigen is presented to the T cell in such a way as to trigger a signal in the T cell via the TCR/CD3 complex. For example, an antigen can be presented to a T cell by an antigen presenting cell in combination with an MHC molecule. An antigen presenting cell, such as a B cell, macrophage, monocyte, dendritic cell, Langerhans cell, or other cell that can present an antigen to a T cell, can be incubated with the T cell in the presence of the antigen (e.g., soluble antigen) such that the antigen presenting the cell presented the antigen to the T cell. Alternatively, a cell expressing an antigen of interest can be incubated with a T cell. For example, a tumor cell expressing tumor-associated antigens can be incubated with a T cell to induce a tumor-specific response. Similarly, a cell infected with a pathogen, such as a virus that presents antigens of the pathogen, can be incubated with a T cell. After antigen-specific activation of the T cell population, the cells can be propagated according to the methods presented. For example, after antigen specificity has been established, T cells can be expanded by culturing with an anti-CD3 antibody (used as the first agent) and an anti-CD28 antibody (used as the second agent) according to the methods described herein. In another embodiment, the first agent may be an MHC I:peptide complex that binds to an antigen-specific population of T cells. In such an embodiment, any known antigen-specific peptide that can complex with the corresponding MHC I molecule can be used. Alternatively, a natural receptor ligand that triggers cell proliferation can also be used as the first agent. For example, the extracellular domain of CD19 can be used to cause the activation of intracellular signaling cascades of cells transduced to express a chimeric CD19 binding antigen receptor (CAR). The above illustrative aspects are shown in the examples.

In some embodiments, a method for culturing a population of cells in vitro is provided, comprising contacting a sample containing a composition containing a plurality of cells with a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents. In some embodiments, the multimerization reagent is a particulate oligomeric reagent. The multimerization reagent and/or particulate oligomeric reagent associated with one or more agents has an agent (first or second, receptor-binding, e.g., stimulatory agent or selective agent) reversibly immobilized thereon that can be used for selection, stimulation, propagation and/or differentiation of cells. In some embodiments, the first agent provides the cells with a primary activation signal, wherein the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents contains at least one Z1 binding site for reversibly binding the first agent. The first agent contains at least one C1 binding partner, wherein the C1 binding partner is capable of reversibly binding to the Z1 binding site of the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents, wherein the first agent is associated with the reagent multimerization via a reversible bond formed between the C1 binding partner and the Z1 binding site. The first agent binds to a receptor molecule on the cell surface, thus providing a primary activation signal to the cells and thus activating the cells.

In some embodiments, the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents is immobilized on a carrier, such as a solid surface. In some embodiments, the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents is not associated with a carrier, eg, not associated with a solid surface or stationary phase.

For example, in some embodiments, a method for propagating a cell population in vitro is provided, comprising contacting a sample containing the cell population with a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, wherein the multimerization reagent and/or the particulate oligomeric reagent associated with one or more agents is not immobilized on a solid support, i. e. It is in soluble form, and has an associated agent (first or second, receptor-binding, for example, stimulating agent or selective agent) that can be used to select, stimulate, expand and/or differentiate cells. In some embodiments, the first agent that provides the cells with a primary activation signal is reversibly linked to a multimerization reagent and/or a particulate oligomeric reagent linked to one or more agents. The multimerization reagent and/or oligomeric reagent in the form of particles associated with one or more agents contains at least one binding site, for example, Z1, for binding the first agent, while the first agent contains at least one binding partner, for example, C1, wherein the C1 binding partner is capable of binding to the Z1 binding site of the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents. In some embodiments, the first agent is associated with the multimerization reagent and/or the particulate oligomeric reagent associated with one or more agents through a bond formed between the C1 binding partner and the Z1 binding site, and the first agent binds to a receptor molecule on the cell surface , thus providing the primary activation signal to the cells and thus activating the cells. In some embodiments, when using a soluble multimerization agent, the relationship between the C binding site, eg, C1, and the Z binding site, eg, Z1, need not be reversible.

For example, in some embodiments, the methods presented also include the use of a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents having a second agent associated therewith, such as additional or costimulatory molecules that stimulate an accessory molecule on the cell surface. . In some cases, the multimerization agent is immobilized on a carrier, such as a solid carrier, solid phase, or stationary phase. In some embodiments, the multimerization agent is not immobilized on the carrier, ie. It is in soluble form. In some embodiments, the second agent comprises a binding partner, such as C2, wherein the binding partner, such as C2, is capable of reversibly binding to a binding site, such as Z2, of a multimerization reagent and/or a particulate oligomeric reagent, wherein the second the agent is linked to the multimerization reagent and/or the particulate oligomeric reagent via a reversible bond formed between the C2 binding partner and the Z2 binding site. In some embodiments, the bond formed between the C1 binding partner and the Z1 binding site may be reversible, and the bond formed between the C2 binding partner and the Z2 binding site may be reversible. In this case, the dissociation constant (Kd) for reversible binding between said binding site Z1 and said binding partner C1 and/or for reversible binding between said binding site Z2 and said binding partner C2 may be in the range from 10 ^-2 M to 10 ^-13 M. In some aspects, for example, when the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents is not associated with a carrier (for example, not associated with a solid carrier or stationary phase), the bond formed between the C1 binding partner and the Z1 binding site may be irreversible, and/or also the bond formed between the C2 binding partner and the Z2 binding site may be irreversible.

In some cases, the second agent binds to a helper molecule on the cell surface, thus stimulating the activated cells. In this embodiment, the first agent may stimulate a TCR/CD3 complex-associated signal in T cells and may be a binding agent that specifically binds CD3. In this embodiment, the accessory molecule on the T cell may be CD28 and the second agent that binds the accessory molecule is a binding reagent that specifically binds CD28. Alternatively, in some embodiments, it has been found that isolation of other additional molecules can also be used, which in some cases can change, for example, improve one or more features, properties or characteristics of cultured cells. In some embodiments, the accessory molecule may be one or more of IL-12R, IFNγR, IL-4R, IL-17R, RORγt, RORα, CXCR3, CCR7, CD62L, CXCR1, or CXCR4 (e.g., anti-IL-12R antibody, anti-IL-12R antibody, IFNγR, anti-IL-4R antibody, and anti-IL-17R antibody, anti-RORγt antibody, anti-RORα antibody, anti-CXCR3 antibody, anti-CCR7 antibody, anti-CD62L antibody, anti-CXCR1 antibody, or anti-CXCR4 antibody, respectively. -binding agents (eg, stimulating agents) are described below.

In some embodiments, the present method can be carried out at any temperature at which the viability of the cell population is at least not significantly affected. In some embodiments, the conditions under which the incubation or culture is carried out include any conditions that are at least not significantly detrimental, do not impair, or at least significantly affect viability, such as where the percentage of the cell population that is desired propagate with full viability is at least 70%, including at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 99.5%. In some embodiments, the implementation of the presented method is carried out at a temperature of approximately 20°C or higher. Depending on the cell population to be expanded, a suitable temperature range may be, for example, from about 20°C to about 45°C, including from about 25°C to about 40°C or from about 32°C to 37°C. C. In some embodiments, the method of the invention is carried out at a constant temperature or at a selectable temperature of ± about 5°C, ± about 4°C, ± about 3°C, ± about 2°C, ± about 1°C, or ± about 0 .5°C. A person skilled in the art is able to empirically determine the appropriate temperature, taking into account the nature of the cells and the propagation conditions. Typically, human cells are propagated at temperatures such as 37°C.

Also provided within the scope of the present invention are multimerization agents or a composition comprising multimerization reagents and/or particulate oligomeric reagents associated with one or more agents that are capable of propagating a population of cells. Such a multimerization agent and/or a particulate oligomeric reagent associated with one or more agents that is capable of propagating a population of cells is a multimerization agent and/or a particulate oligomeric reagent associated with one or more agents that is not associated with a carrier ( for example, in soluble form) and contains at least one Z binding site, for example, Z1, for reversible binding of the first agent that provides the cells with a primary activation signal, while the multimerization reagent and / or oligomeric reagent in the form of particles associated with one or more agents, has said first agent reversibly associated with it, which provides the cells with a primary activation signal; wherein the first agent comprises at least one C binding partner, e.g., C1, wherein the C1 binding partner is capable of reversibly binding to at least one Z1 binding site of the multimerization reagent and/or particulate oligomeric reagent associated with one or more agents, wherein the first agent is linked to a multimerization reagent and/or a particulate oligomeric reagent linked to one or more agents via a reversible bond formed between a C1 binding partner and a binding site Z1. In this case, it should be noted that such a multimerization agent may have immobilized on it any of the first agents that are described in this document.

In some embodiments, the multimerization reagent provided herein may further comprise at least one binding site, such as Z2, for reversibly binding a second agent that stimulates an accessory molecule on the cell surface, wherein the multimerization reagent and/or oligomeric reagent in particulate, associated with one or more agents, has a second agent reversibly associated with it, which stimulates an auxiliary molecule on the surface of cells, while the second agent contains a binding partner, for example, C2, while the C2 binding partner is able to bind at least with one site Z2 binding of the multimerization reagent and/or oligomeric reagent in the form of particles. In this embodiment, the second agent is linked to the multimerization reagent and/or the particulate oligomeric reagent via a bond formed between the C2 binding partner and the Z2 binding site. In some embodiments, the second agent is any that can bind to IL-12R, IFNγR, IL-4R, IL-17R, RORγt, RORα, CXCR3, CCR7, CD62L, CXCR1, or CXCR4 (e.g., anti-IL-12R antibody, antibody anti-IFNγR, anti-IL-4R, and anti-IL-17R, anti-RORγt, anti-RORα, anti-CXCR3, anti-CCR7, anti-CD62L, anti-CXCR1, or anti-CXCR4, respectively).

In some embodiments, culturing a composition comprising target cells (e.g., T cells) with a multimerized agent (e.g., an anti-CD3/anti-CD28 streptavidin mutein or oligomer thereof) can be performed in a bioreactor, such as a hollow fiber bioreactor (e.g., a hollow fiber bioreactor of the Quantum® cell expansion) or a bioreactor with a plastic container (such as the Cellbag® used in the Xuri Cell Expansion System W25 from GE Healthcare).

In some embodiments, the method further comprises contacting cultured target cells (e.g., T cells) in a reaction mixture (e.g., containing target cells, e.g., T cells associated with a multimerization reagent and/or a particulate oligomeric reagent , for example, through the first agent and the second agent) with (i) a competitive reagent (for example, a free first binding partner C, for example, C1) or its analogue, capable of breaking the link between the first binding partner, for example, C1, and the binding site , for example, Z1, and/or (for example, if necessary) (ii) a second competitive reagent, for example, a free second binding partner, for example, C2, or an analogue thereof, capable of breaking the link between the second C2 binding partner and the Z2 binding site . This breaks the reversible bond between said binding partner C1 of the first agent and said binding sites Z1, as well as the reversible bond between said binding partner C2 of the second agent and said binding site Z2 of said multimerization reagent and/or particulate oligomeric reagent, such thereby releasing in the eluate the T cells bound to the multimerization reagent and/or the particulate oligomer reagent via the first agent and the second agent, and disrupting T cell stimulation and/or expansion.

In some embodiments, a competitive reagent (e.g., the first and/or second competitive reagent) is added within 14 days, 10 days, 7 days, or 5 days after the start of incubation, e.g., within 10 days, 9 days, 8 days, 7 days , 6 days, 5 days, 4 days, 3 days, 2 days or 1 day after the start of incubation. In specific embodiments, the implementation of the competitive reagent is added within 5 days after the start of incubation, for example, within 4 days, 3 days, 2 days or 1 day after the start of incubation. In some embodiments, the competitive reagent is added within 1 day of the start of incubation, such as within 18 hours, 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 90 minutes, 60 minutes, or 30 minutes after the start of incubation. Therefore, by adjusting the time at which stimulation is interrupted, one or more distinct features of the cultured T cells eluted from the multimerized agent can be altered, as described herein. For example, in some embodiments, if the addition of a competitive reagent is started within 5 days, 4 days, 3 days, 2 days, or 1 day after incubation, it will increase stimulation, activation, enrichment, expansion, selection, and/or proliferation of one or more cultured cells.

In some embodiments, the implementation of the method further includes the separation or removal of one or more components remaining after the reversible dissociation of the components. In some embodiments, any unbound or residual biotin in cultured target cells (eg, T cells) can be separated or removed. In some embodiments, the multimerization reagent and/or the particulate oligomeric reagent are removed or separated from cells in the cultured target cell composition. For example, in some embodiments, separation/removal can be performed using a second stationary phase. For this purpose, a mixture containing target cells (for example, T cells) and a soluble multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, before or after application to the first stationary phase described above, is subjected to chromatography. on a suitable second stationary phase. This secondary stationary phase may be a gel filtration matrix and/or an affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix contains an affinity reagent. The affinity reagent, consisting of a chromatographic resin, includes a binding partner D that (specifically) binds to the Z1 binding site and/or the Z2 binding site, if present, a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, thereby immobilizing on the stationary phase a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents. if a streptavidin-based multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents is used, and the first and second agents have a streptavidin-binding peptide as binding partner C1 or C2, binding partner D which is contained in the affinity reagent of this second stationary phase may be biotin. The soluble streptavidin oligomer or streptavidin mutein that is used as the multimerization and/or particulate oligomer reagent is then coupled to biotin, which is typically covalently attached to a chromatography matrix such as the commercially available Biotin-Sepharose™. In some such embodiments, cultured cells (eg, cultured T cells) can be recovered from the multimerization reagent and/or particulate oligomeric reagent.

A. Cells

The cells contained in the target cell composition are usually eukaryotic cells, such as mammalian cells, and are usually human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, or they are cells of the immune system, such as cells of innate or adaptive immunity, for example, myeloid or lymphoid cells, including lymphocytes, usually T cells and/or NK -cells. Other exemplary cells include stem cells such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). Cells are typically primary cells, such as cells isolated directly from the subject and/or isolated from the subject and frozen.

In some embodiments, reversibly coupled agents, such as multimerized agents provided herein, are capable of propagating a population of lymphocytes or a subpopulation contained within a population of lymphocytes. The population of lymphocytes to be expanded may be any suitable population, for example, a population of B cells, a population of T cells, or a population of natural killer cells. The T cell population may be an antigen-specific T cell population, a helper T cell population, a cytotoxic T cell population, a memory T cell population, a regulatory T cell population, or a natural killer T cell population. Accordingly, in such embodiments of the multimerized reagent, the first agent is capable of stimulating a TCR/CD3 complex-associated signal in T cells. The first agent present in the multimerized agent may thus be a binding agent that specifically binds CD3, while the second agent that binds the accessory molecule, for example, may be a binding agent that specifically binds CD28, CD137, IL-12R, IFNγR, IL-4R, IL-17R, RORγt, RORα, CXCR3, CCR7, CD62L, CXCR1, or CXCR4.

In some embodiments, the cells include one or more subsets of T cells or other cell types, such as all T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as subpopulations defined by function, activation state, maturity, potential for ability differentiation, multiplication, recycling, localization and/or persistence, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, cytokine secretion marker or profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Methods include standard methods. In some aspects, eg for standard technologies, the cells are pluripotent and/or multipotent, eg stem cells such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, obtaining, processing, culturing and/or constructing them as described herein, and reintroducing them to the same patient before or after cryopreservation.

Among the subtypes and subpopulations of T cells and/or CD4+ and/or CD8+ T cells are naive (T _N ) T cells, effector T cells (T _EFF ), memory T cells and their subtypes, such as stem T- memory cells (T _SCM ), central memory T cells (T _CM ), effector memory T cells (T _EM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells cells, T helpers, cytotoxic T cells, mucosal associated invariant T cells (MAIT), naturally occurring and adaptive regulatory T cells (Treg), T helpers such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.

In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.

1. Obtaining cells

In some embodiments, obtaining cells includes one or more stages of culturing and/or obtaining. Cells can be isolated from a sample, such as a biological sample, for example, a sample originating from or obtained from a subject. In some embodiments, the subject from which the cells are isolated is a subject who has a disease or condition or is in need of cell therapy or for whom cell therapy will be used. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for which cells are isolated, processed and/or engineered.

Accordingly, the cells in some embodiments are primary cells, such as primary human cells. Samples include tissue, fluid, and other samples taken directly from the subject, as well as samples obtained from one or more processing steps such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and/or incubation . the biological sample may be a sample obtained directly from a biological source or a processed sample. biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived from them.

In some aspects, the sample from which cells are obtained or isolated is blood, or a blood-derived sample, or is, or is obtained from an apheresis or leukapheresis product. Illustrative specimens include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bones, prostate, cervix, testicles, ovaries, tonsils or other organs and/or cells derived from them. In the context of cell therapy, such as adoptive cell therapy, samples include samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, such as T cell lines. The cells, in some embodiments, are derived from a xenogeneic source, such as a mouse, rat, non-human primate, or pig.

In some embodiments, cell isolation includes one or more steps of obtaining and/or separating cells based on lack of affinity. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich with desired components, lyse, or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more properties such as density, adhesive properties, size, sensitivity, and/or resistance to particular components.

In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. In some aspects, the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and/or platelets, and in some aspects contain cells other than red blood cells and platelets.

In some embodiments, blood cells taken from a subject are washed, for example, to remove a plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all of the divalent cations. In some aspects, the washing step is performed using a semi-automated "flow" centrifuge (eg, Cobe 2991 cell processor, Baxter) in accordance with the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are resuspended in a variety of biocompatible buffers, such as, for example, Ca ⁺⁺ /Mg ⁺⁺ free PBS. In some embodiments, components of the blood cell samples are removed and the cells directly resuspended in culture medium.

In some embodiments, the methods include methods for separating cells based on density, such as obtaining white blood cells from peripheral blood by lysing the red blood cells and centrifuging through a Percoll or Ficoll gradient.

In some embodiments, isolation methods include separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, such as surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known separation method based on such markers can be used. Separation methods may include any of the methods disclosed herein, including methods using reversible systems of reagents, for example, agents (such as receptor-binding agents or selective agents) and reagents, which are described in this document.

In some embodiments, the separation is based on affinity or immunoaffinity. For example, isolation in some aspects involves separating cells and cell populations based on cellular expression or the level of expression of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, usually followed by the steps of washing and separating cells having a bound antibody or binding partner from those cells that are not bound to the antibody or binding partner.

Such separation steps may be based on positive selection, in which cells having bound reagents are retained for further use, and/or negative selection, in which cells not bound to the antibody or binding partner are retained. In some examples, both fractions are left for further use. In some aspects, negative selection can be particularly useful when no antibody is available that specifically identifies a cell type in a heterogeneous population in such a way as to best distinguish based on markers expressed by cells that are not in the desired population.

Separation should not lead to 100% enrichment or removal of a particular population of cells or cells expressing a particular marker. For example, positive selection or enrichment for a particular type of cell, such as marker-expressing cells, refers to an increase in the number or percentage of such cells, but does not necessarily lead to a complete absence of cells not expressing the marker. Also, negative selection, removal or reduction of cells of a particular type, such as cells expressing a marker, refers to a decrease in the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

In some examples, multiple cycles of separation steps are performed where a positively or negatively selected fraction from one stage is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, cells expressing multiple markers simultaneously can be eliminated in a single separation step, for example, by incubating the cells with multiple antibodies or binding partners, each of which is specific for the marker isolated for negative selection. Also, multiple cell types can be simultaneously positively selected by incubating cells with multiple antibodies or binding partners expressed on different cell types.

For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, for example, CD28 ⁺ , CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD127 ⁺ , CD4 ⁺ , CD8 ⁺ , CD45RA ⁺ and/or CD45RO ⁺ T cells are isolated using positive or negative selection techniques.

For example, positive selection for CD3 ⁺ , CD28 ⁺ T cells can be performed using CD3/CD28 conjugated magnetic beads (eg DYNABEADS® M-450 CD3/CD28 T cell culture device).

In some embodiments, selections are made by enriching a particular population of cells using positive selection or depleting a specific population of cells using negative selection. In some embodiments, positive or negative selection is performed by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker ⁺ ) at a relatively higher level (marker ^high ) at a positive or negatively selected cells, respectively.

In some embodiments, T cells are isolated from a PBMC sample by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells such as CD14. In some aspects, a CD4 ⁺ or CD8 ⁺ selection step is used to separate CD4 ⁺ helper and CD8 ⁺ cytotoxic T cells. Such CD4 ⁺ and CD8 ⁺ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on one or more subpopulations of naive, memory and/or effector T cells.

In some embodiments, CD8 ⁺ cells are further enriched or depleted in naïve, memory central, memory effector, and/or central memory stem cells, eg, by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment in central memory T cells (T _CM ) is performed to increase efficiency, for example, to improve long-term survival, expansion and/or engraftment after administration, which in some aspects is especially large in such subpopulations. See Terakuraet al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, the combination of TCM-enriched _CD8 + T cells and CD4+ T cells further enhances efficacy.

In embodiments, memory T cells are present in both CD62L ⁺ and CD62L ^- subgroups of CD8 ⁺ peripheral blood lymphocytes. PBMC can be enriched or depleted in CD62L ^- CD8 ⁺ and/or CD62L ⁺ CD8 ⁺ fractions, for example using anti-CD8 and anti-CD62L antibodies.

In some embodiments, central memory T-cell (TCM) enrichment is based on positive or high expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127 on the surface; in some aspects it is based on negative selection for cells expressing or strongly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8 ⁺ population enriched in T _CM cells is accomplished by depletion of cells expressing CD4, CD14, CD45RA and positive selection or enrichment in cells expressing CD62L. In one aspect, central memory T cell (TCM) enrichment is performed by starting with a negative fraction of cells selected based on CD4 expression, which are negatively selected based on CD14 and CD45RA expression, and positively selected based on CD62L. Such selections are in some aspects carried out simultaneously, and in other aspects are carried out sequentially, in any order. In some aspects, the same CD4 expression-based selection step used to generate a CD8 ⁺ population or subset of cells is also used to generate a CD4 ⁺ population or subset of cells such that both the positive and negative fractions from the CD4-based separation are retained and used. in subsequent steps of the methods, optionally after one or more additional positive or negative selection steps.

CD4 ⁺ T helpers are sorted into naive, central memory and effector cells by identifying a population of cells that have cell surface antigens. CD4 ⁺ lymphocytes can be obtained using standard methods. In some embodiments, naïve CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, the central memory cells of CD4 ⁺ are CD62L ⁺ and CD45RO ⁺ . In some embodiments, the CD4 ⁺ effector cells ^are CD62L- and ^CD45RO-

In one example, for CD4 ⁺ cell enrichment by negative selection, the mixture of monoclonal antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is coupled to a solid support or matrix, such as a magnetic bead or paramagnetic bead, allowing cells to be separated for positive and/or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (discussed in the methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: SA Brooks and U. Schumacher © Humana Press Inc. Totowa, NJ).

In some aspects, the sample or cell composition to be separated is incubated with a small, magnetizable or magnetizable material, such as magnetizable particles or microparticles such as paramagnetic beads (eg, such as Dynalbeads or MACS). The magnetization-responsive material, such as particles, is usually directly or indirectly associated with a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on the cell, cells, or cell population to be separated, such as which must be subjected to negative or positive selection.

In some embodiments, the magnetic particle or bead comprises a magnetization-responsive material associated with a specific binding element, such as an antibody or other binding partner. There are many well known magnetization responsive materials used in magnetic separation processes. Suitable magnetic particles include those described in Molday US Pat. No. 4,452,773 and European Patent Specification EP 452342 B, which are incorporated herein by reference. Other examples are colloidal sized particles such as those described by Owen US Pat. No. 4,795,698 and Liberti et al. US Pat. No. 5,200,084.

The incubation is usually carried out under conditions in which antibodies or binding partners or molecules, such as secondary antibodies or other reagents, that specifically bind to such antibodies or binding partners, which are associated with a magnetic particle or bead, if present on cells in the sample, bind specifically to cell surface molecules.

In some aspects, the sample is placed in a magnetic field, and those cells that have magnetization-responsive or magnetizable particles attached to them will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells are left that are attracted to the magnet; for negative selection, cells that are not attracted (unlabeled cells) are left. In some aspects, during the same selection stage, a combination of positive and negative selection is carried out, where positive and negative fractions are left and further processed or subjected to additional separation steps.

In some embodiments, magnetization-responsive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In some embodiments, magnetic particles are attached to cells by coating with primary antibodies specific for one or more markers. In some embodiments, primary antibodies or binding partner label cells rather than beads, and then add magnetic particles coated with secondary antibodies or other binding partner (eg, streptavidin) specific to that cell type. In some embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies.

In some embodiments, magnetization-responsive particles are left attached to cultured and/or engineered cells, which subsequently need to be incubated; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, magnetizable or magnetizable particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

In some embodiments, affinity-based selection is performed by magnetically activated cell sorting (MACS) (Miltenyi Biotech, Auburn, CA). Magnetically Activated Cell Sorting Systems (MACS) are capable of selecting cells having magnetized particles attached to them with high purity. In some embodiments, MACS operates in a mode where non-target and target particles are sequentially eluted after application of an external magnetic field, i.e., cells attached to magnetized particles are held in place while unattached particles are eluted. Then, after this first elution step is completed, the particles that were trapped in the magnetic field and prevented from eluting are somehow released so that they can be eluted and recovered. In some embodiments, non-target cells are labeled and removed from a heterogeneous population of cells.

In some embodiments, the isolation or separation is carried out using a system, device, or apparatus that performs one or more of the steps of the methods for isolating, obtaining, separating, processing, incubating, culturing, and/or obtaining a finished form of cells. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize error, user handling, and/or contamination. In one example, the system is the system as described in International Patent Application Publication Number WO 2009/072003 or US 20110003380 A1.

In some embodiments, the system or device performs one or more, for example, all of the steps of isolation, processing, design, and shaping in an integrated or stand-alone system and/or in automatic or programmable mode. In some aspects, the system or device includes a computer and/or a computer program that interacts with the system or device that allows a user to program, control, evaluate, and/or regulate various aspects of the processing, isolation, construction, and shaping steps.

In some aspects, the separation steps and/or others are carried out using the CliniMACS system (Miltenyi Biotic), for example, to automatically separate cells at the clinical scale level in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various shut-off valves. In some aspects, an integrated computer controls all components of the instrument and directs the system to perform repetitive techniques in a standardized sequence. the magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the sampling column. The peristaltic pump controls the flow rate throughout the entire set of tubes and, together with check valves, provides a controlled flow of buffer through the system and a continuous cell suspension.

in some aspects, the CliniMACS system uses antibody-bound magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. the cell collection bag is then connected to a set of tubes, which in turn are connected to a bag containing the buffer and the cell collection bag. The tubing set consists of pre-assembled sterile tubing, including guard column and separation column, for one use only. Once the separation program has started, the system automatically places the cell sample onto the separation column. Labeled cells are left inside the column, while unlabeled cells are removed using a series of washing steps. In some embodiments, cell populations for use with the methods described herein are unlabeled and do not remain on the column. In some embodiments, cell populations for use with the methods described herein are labeled and remain on the column. In some embodiments, cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and collected in a bag to obtain cells.

In some embodiments, the separation steps and/or others are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some aspects, the CliniMACS Prodigy system is equipped with a cell processing unit that provides automatic washing and cell fractionation by centrifugation. The CliniMACS Prodigy system can also include an integrated camera and image recognition software that determines the optimal cell fractionation endpoint by recognizing macroscopic product layers with parent cells. For example, peripheral blood can be automatically separated into layers of red blood cells, white blood cells, and plasma. The CliniMACS Prodigy system can also contain an integrated cell culture chamber that performs cell culture protocols such as, for example, cell differentiation and expansion, antigen loading, and long-term cell culture. Inlet ports can allow for sterile removal and replenishment of media, and cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, the cell population described herein is harvested and enriched (or depleted) by flow cytometry, in which cells stained for a plurality of cell surface markers are transferred in a fluid stream. In some embodiments, a population of cells described herein is harvested and enriched (or depleted) by preparative sorting (FACS). In some embodiments, the population of cells described herein is harvested and enriched (or depleted) through the use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, for example, WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573 and Godin et al (2008) J Biophoton 1(5):355-376 In both cases, cells can be labeled with a variety of markers, allowing the isolation of well-defined subgroups of T cells with high purity.

In some embodiments, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, cell separation based on the binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid stream, for example, using fluorescence-activated cell sorting (FACS), including preparative (FACS) chips and/or or microelectromechanical systems (MEMS), for example, in combination with a detection system by flow cytometry. Such methods involve positive and negative selection simultaneously based on multiple markers.

In some embodiments, the preparation methods include the steps of freezing, eg, cryopreserving the cells, either before or after isolation, incubation, and/or construction. In some embodiments, granulocytes and, to some extent, monocytes are removed from the cell population during the freezing and subsequent thawing step. In some embodiments, the cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves the use of PBS containing 20% DMSO and 8% human serum albumin (HSA) or other suitable cell freezing medium. it is then diluted 1:1 with the medium so that the final concentration of DMSO and HSA is 10% and 4%, respectively. the cells are then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

In some embodiments, cells are incubated and/or cultured prior to or in conjunction with genetic engineering. The incubation steps may include culturing, growing, stimulating, activating and/or propagating. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include conditions designed to induce proliferation, expansion, activation and/or survival of cells in a population, to mimic antigen exposure, and/or to prime cells for genetic engineering, for example, to introduce a recombinant antigen receptor.

Conditions may include one or more of specific environment, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors such as cytokines, chemokines, antigens, binding partners. , fusion proteins, recombinant soluble receptors, and any other agents designed to activate cells.

In some embodiments, the stimulation conditions or agents include one or more agents, such as a ligand, that is capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates an intracellular TCR/CD3 signaling cascade in a T cell. Such agents may include antibodies, such as antibodies specific for a TCR component and/or a costimulatory receptor, eg anti-CD3, anti-CD28, eg associated with a solid carrier such as a bead, and/or one or more cytokines. Optionally, the propagation method may further comprise the step of adding an anti-CD3 and/or anti-CD28 antibody (eg, at a concentration of at least about 0.5 pg/mL) to the culture medium. In some embodiments, the stimulatory agents include IL-2 and/or IL-15, for example, the concentration of IL-2 is at least about 10 units/ml.

In some aspects, the incubation is carried out in accordance with techniques such as those described in US patent No. 60401 77 Riddell et al. Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.

In some embodiments, T cells are expanded by adding nurse cells, such as non-profiling peripheral blood mononuclear cells (PBMC) to the composition (e.g., such that the resulting cell population contains at least about 5, 10, 20, or 40 or more nurse cells). PBMC cells for each T-lymphocyte in the initial population to be expanded); and culture incubation (eg, for a period of time sufficient to expand a number of T cells). In some aspects, non-profiling nurses may include gamma irradiated PBMC nurses. In some embodiments, the implementation of PBMC irradiated with gamma radiation in the range from about 3000 to 3600 rad to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of the T cell population.

In some embodiments, stimulation conditions include a temperature suitable for growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees, and typically at or about 37 degrees Celsius. Optionally, the incubation may further include the addition of non-profiling EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma radiation in the range from about 6000 to 10000 rad. In some aspects, LCL feeder cells are provided in any suitable amount, for example, in a ratio of LCL feeder cells to primary T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by antigen stimulation of naive or antigen-specific T lymphocytes. For example, antigen-specific T cell lines or clones can be generated for cytomegavirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.

B. Device and Manufacturers' Products

In some embodiments, a device or finished product is also provided. In some embodiments, a bioreactor device and a first chromatographic stationary phase are provided. the bioreactor is suitable for cell expansion, and the stationary phase is suitable for cell separation and removal of reagents. The first stationary phase is a gel filtration matrix and/or an affinity chromatography matrix, while the gel filtration and/or affinity chromatography matrix contains an affinity reagent, while the affinity reagent contains a binding site Z1 that specifically binds to a binding partner C1 contained in the first agent, and/or the affinity reagent contains a Z2 binding site that specifically binds to a C2 binding partner contained in the second agent. The first stationary phase is thus suitable for immobilizing thereon the first agent and/or the second agent, the first binding partner C1 and/or the free second binding partner C2. In addition, the bioreactor and the stationary phase are connected with the possibility of passing the fluid. This device can be used for serial expansion as explained above and can be integrated into known cell expansion systems such as the Quantum® cell expansion system) or the W25 Xuri cell expansion system.

In this device, the first stationary phase is either contained in a chromatographic column or is a planar stationary phase. The device may further comprise a second stationary phase that is fluidly connected to the first stationary phase. The secondary stationary phase may be a gel filtration matrix and/or an affinity chromatography matrix, wherein the gel filtration and/or affinity chromatography matrix contains an affinity reagent. This affinity reagent may contain a binding partner D that (specifically) binds to the Z1 binding site of the multimerization reagent and/or particulate oligomeric reagent and is thus suitable for immobilizing the multimerization reagent and/or particulate oligomeric reagent on the stationary phase.

In addition, the invention is directed to a device for purification (for example, selection) and cultivation, such as stimulation or propagation, of a composition of cells, and the device contains at least one bioreactor device and a first stationary phase and/or a second stationary phase for chromatography, as defined above.

The device may further comprise a plurality of bioreactor devices and a stationary phase connected in series to allow fluid to pass through.

The device may include a sample inlet in fluid communication with the bioreactor of the bioreactor device and the chromatography stationary phase. The apparatus may also comprise a sample outlet for purified and expanded target cells, wherein the sample outlet is connected to allow passage of the stationary phase fluid of the last of the at least one bioreactor device and the chromatography stationary phase.

In some embodiments, the implementation of the device may be designed as a functionally closed system.

C. Exemplary Characteristics of Cultured Cells

In some embodiments, cultured target cells (e.g., cultured T cells), which may include cultured cells generated or obtained according to the methods provided herein, exhibit one or more of the indicated phenotypic and/or functional features based on or associated with their ability to proliferate, surface marker expression, differentiation state, activation state, and/or metabolic profile. In some embodiments, culturing target cells (e.g., culturing T cells) according to any of the present methods results in a change in a parameter associated with function (e.g., increased or decreased functional activity) or phenotype (e.g., higher or lower expression of the marker or markers) of cells compared to a similar or corresponding cell function or phenotype in a composition prior to incubation according to the methods provided herein. In some embodiments, the cultured T cells exhibit a change in a parameter of replication and/or proliferation, distribution or ratio of CD4+/CD8+ T cells, surface marker expression, functional activity, or metabolic profile.

In some embodiments, the change in a parameter as measured in cultured T cells is compared or contrasted with the same or similar parameter as measured in a reference composition or T cell preparation. Typically, T cells in a reference composition or T cell preparation include or are derived from the same or substantially the same composition of T cells before incubation with a reversibly coupled agent (e.g., a multimerized agent, e.g., before incubation with a stimulatory agent reversibly streptavidin oligomeric mutein), except that such cells were not incubated or otherwise incubated. In some embodiments, a reference T cell preparation is incubated using substantially the same protocol or conditions (e.g., type of stimulating agents or agent, format of stimulating agents or agent, substantially the same amounts of starting cells, washes, presence or absence of additional reagents, incubation timing, incubation temperature) except that at least one aspect, and in some cases only one aspect, of such incubation in a T cell reference preparation differs from incubation to produce cultured T cells.

In some embodiments, the reference composition or T cell preparation is a composition containing T cells prior to incubation with a reversibly bound agent (e.g., a multimerized agent, such as prior to incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein).

In some embodiments, cultured T cells are generated by incubation with a reversibly coupled agent (e.g., a multimerized agent, e.g., incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein) for less than 28 days, 21 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day and/or when the association of such an agent with one or more molecules on the cell is disrupted (for example, in the presence of a competitive reagent, for example , biotin or biotin analogue), for example, is broken in 28 days, 21 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day from start incubation with such an agent. For example, in some aspects, cultured T cells are generated or obtained after incubation with a reversibly coupled agent (e.g., a multimerized agent, e.g., after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein) as described herein, and the incubation is terminated and/or violate within 28 days, 21 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days after the start of such incubation (for example, within or about 4, 3, 2 or 1 days or less), and/or when a competing agent (e.g., biotin) that dissociates the reversibly bound agent from cells is added to incubated cells for 28 days, 21 days, 14 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days after the start of such incubation (eg, within or about 4, 3, 2, or 1 day or less). In some embodiments, a reference T cell preparation is made or obtained after incubation with the same or substantially the same reversibly coupled agent (e.g., a multimerized agent, e.g., after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), but where the incubation is performed for more than 5 days, do not stop and/or disrupt to attenuate or stop the signal induced or modulated in the cell, and/or where the T cell preparation is obtained without the addition of a competing agent (for example, biotin or a biotin analog) that dissociates the reagent from cells.

In some embodiments, cultured T cells are generated by incubation with a reversibly bound agent (e.g., a multimerized agent, e.g., incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), wherein the receptor-binding agent (e.g., stimulatory agent) is an agent, that does not bind to CD28 and/or cause signaling, ie. Not an anti-CD28 antibody or a fragment thereof. For example, in some embodiments, cultured T cells are obtained or created after incubation with a reversibly coupled reagent, wherein one or more stimulatory agents reversibly bind to a streptavidin mutein, wherein at least one stimulatory agent is specific for CD3 (e.g., an anti-CD3 antibody). or a fragment thereof) and the second stimulatory agent may be specific for one or more of CD90, CD95, CD137, CD154, ICOS, LAT, CD27, OX40, or HVEM (e.g., anti-CD90, anti-CD95, anti-CD137, and anti-CD154, anti-ICOS, anti-LAT, anti-CD27, anti-OX40, or anti-HVEM, respectively, or antigen-binding fragments thereof). In some embodiments, the reference T cell preparation is a T cell culture created or obtained after incubation with a reversibly linked agent (e.g., a multimerized agent, e.g., after incubation with a stimulatory agent reversibly linked to a streptavidin oligomeric mutein), but where the reagent contains an agent that specifically binds CD28 and/or induces or modulates CD28 signaling. For example, in some embodiments, a reference T cell preparation is created or obtained after incubation of a T cell composition with anti-CD3/anti-CD28 Dynabeads®, anti-CD3/anti-CD28 ExPact® beads, or other anti-CD3/anti-CD28 stimulatory agent. In some embodiments, such another anti-CD3/anti-CD28 stimulatory agent is an agent in which antibody reagents are bound to a carrier (e.g., a solid carrier), e.g., bead, particles, magnetic particles or bead, nanoparticles, or microsphere. In some embodiments, cultured T cells are obtained by incubation with a reversibly bound agent (e.g., a multimerized agent, e.g., incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein) that is soluble, i. Not associated with a carrier (for example, a solid carrier).

For example, in some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are generated or obtained after incubation with a reversibly linked agent as described herein (for example, multimerized agent, e.g. after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), the cultured T cells having an improved ability to proliferate and/or proliferate compared to a reference T cell composition or preparation. In some embodiments, the improved ability to proliferate and/or proliferate includes increasing the number or percentage of CD3+ T cells, CD4+ T cells, and/or CD8+ T cells in cultured T cells by at least about 2-fold (e.g., , any of at least about 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) compared to the number or percentage of CD3+ T cells, CD4+ T cells and/or CD8+ T cells, respectively, in a reference composition or T cell preparation.

In some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are created or obtained after incubation with a reversibly linked agent, as described herein (for example, a multimerized agent, for example, after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), cultured T cells having improved ability to expand and/or proliferate CD3+ T cells compared to a reference T cell culture. In some embodiments, the improved ability to proliferate and/or proliferate includes increasing the number or percentage of CD3+ T cells in cultured T cells by at least about 2-fold (e.g., any of at least about 3-fold, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) compared to the number or percentage of CD3+ T cells in a reference composition or T cell preparation.

In some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are created or obtained after incubation with a reversibly linked agent, as described herein (for example, a multimerized agent, e.g., after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), cultured T cells having an improved ability to expand and/or proliferate CD4+ T cells compared to a reference T cell composition or preparation. In some embodiments, the improved ability to proliferate and/or proliferate includes increasing the number or percentage of CD4+ T cells in cultured T cells by at least about 2-fold (e.g., any of at least about 3-fold, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) compared to the number or percentage of CD4+ T cells in a reference composition or T cell preparation.

In some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are created or obtained after incubation with a reversibly linked agent, as described herein (for example, a multimerized agent, e.g. after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), cultured T cells having improved expansion and/or proliferation of CD8+ T cells compared to a reference T cell composition or preparation. In some embodiments, the improved ability to proliferate and/or proliferate includes increasing the number or percentage of CD8+ T cells in cultured T cells by at least about 2-fold (e.g., any of at least about 3-fold, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more) compared to the number or percentage of CD8+ T cells in a reference composition or T cell preparation.

In some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are created or obtained after incubation with a reversibly linked agent, as described herein (for example, a multimerized agent, e.g. after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), the cultured T cells being characterized by an altered distribution of CD8+/CD4+ T cells or a normalized distribution of T cells, e.g. an altered ratio of CD8+/CD4+ T cells or a normalized ratio CD8+/CD4+ T cells compared to a reference composition or T cell preparation. The CD8+/CD4+ ratio or normalized ratio can be increased or decreased. In some embodiments, the altered ratio of CD8+/CD4+ T cells is due to an increase in the number or percentage or normalized number or percentage of CD8+ T cells in cultured T cells relative to or compared to the number or percentage or normalized number or percentage in a reference composition or preparation. In some embodiments, the number of CD8+ T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of CD8+ T cells or the normalized number or percentage of CD8+ T cells in a reference composition or T cell preparation. In some embodiments, the CD8+/CD4+ T cell ratio or normalized CD8+/CD4+ ratio is increased at least about 2-fold (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 times, 9 times, 10 times or more) compared to the CD8+/CD4+ T cell ratio or the normalized CD8+/CD4+ ratio in the reference composition or T cell preparation. In some embodiments, the number, percentage, or ratio of cultured T cells or in the composition or preparation is normalized to the number, percentage, or ratio in the original composition containing T cells prior to incubation.

In some embodiments, cultured T cells are provided according to any of the methods provided herein, wherein the cultured T cells are generated or obtained after incubation with a reversibly linked agent as described herein (e.g., a multimerized agent, e.g., after incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein), and the cultured T cells show an altered expression profile of the surface marker compared to the reference composition or T cell preparation. In some embodiments, the altered surface marker expression profile is due to a change in the number or percentage of one or more T cell subgroups that are positive, negative, or low for one or more surface markers selected from CD45RA, CD45RO, CD62L, CD69, CCR7, CD27, CD28, CD122, t-bet, IL-7Rα, CD95, IL-2Rβ, CXCR3, LFA-1, KLRG1. In some embodiments, the number or percentage of a subset of T cells in cultured T cells is increased by at least about 2-fold (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8 times, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or preparation.

In some embodiments, a subset of T cells in cultured T cells (e.g., a subset of T cells that is increased in cultured T cells compared to a reference composition or preparation) exhibits a state of reduced or diminished differentiation or activation compared to a reference composition, or T cell preparation. In some embodiments, the subset of T cells is not or does not contain the effector T cell ( _TE ) or memory effector T cell (T _EM ) phenotype. In some embodiments, the subset of T cells contains a surface phenotype that is one or more of CD62L ⁺ , CCR7 ⁺ , CD27 ⁺ , CD28 ⁺ , or KLRG1 ^low/ . In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, a subset of T cells in cultured T cells (e.g., a subset of T cells that is increased in cultured T cells compared to a reference composition or formulation) is positive for CD62L and/or IL-7Rα (CD127) and /or negative or low for t-bet. In some embodiments, the T cell subset is positive for CD45RA and/or negative or low for CD45RO. In some embodiments, a subset of T cells is positive for one or more of CCR7, CD45RA, CD62L, CD27, CD28, IL-7Rα (CD127), CD95, IL-2Rβ, CXCR3, and LFA-1 and/or negative for CD45RO. In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, the subset of T cells in cultured T cells (e.g., the subset of T cells that is increased in cultured T cells compared to a reference composition or preparation) is or contains cells that are positive for CD62L (CD62L+). In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, a subset of T cells in cultured T cells (e.g., a subset of T cells that is increased in cultured T cells compared to a reference composition or preparation) is or contains cells that are CD62L+ and a) any one or more of CD45RA ^low/+ , CD45RO ^low/+ , CCR7+ and CD27+ and b) any one or more of t-bet ^low , IL-7Rα+ (CD127+), CD95+, IL-2Rβ+, CXCR3+ and LFA-1+ . In some embodiments, the subset of T cells can also be CD3+, CD4+, or CD8+. In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, a subset of T cells, such as a subset of CD62L+ T cells, in cultured T cells are or contain or share phenotypic characteristics with memory T cells or specific subgroups thereof, such as long-lived memory T cells. In some embodiments, such memory T cells are central memory T cells (Tcm) or stem memory T cells (Tscm). In some embodiments, the memory T cells are Tscm cells. Tscm cells can be described as having one or more phenotypic differences or functional traits compared to other subgroups of memory T cells or compared to naive T cells, e.g., less differentiated or more naive (see, e.g., Ahlers and Belyakov (2010) Blood 115:1678); Cieri et al. (2015) Blood. 125:2865; Flynn et al. (2014) Clinical & Translational Immunology, 3, e20; Gattinoni et al. (2012) Nat. Med. 17:1290-1297; Gattinoni et al. (2012) Nat. Reviews, 12:671; Li et al. (2013) PLOS ONE, 8:e67401; and PCT Publication No. WO 2014/039044). In some cases, it is believed that Tscm cells are only memory T cells capable of creating effector T cells and all three subsets of memory T cells (Tscm, Tcm, and Tem). In some aspects, Tscm cells have the highest survival and proliferation response to antigenic or homeostatic stimuli of all memory T cell subgroups and the least depletion in the absence of cognate antigen. In some embodiments, less differentiated Tscm cells may exhibit greater expansion, long-term viability, and target cell killing after adoptive transfer than other memory T cells, and thus may be able to mediate more effective treatment with fewer transferred cells than would be possible for either Tcm or Tem cells.

In some aspects, examples of phenotypic or functional features reported or known for Tscm cells include, for example, that such cells a) are CD45RO ^- , CCR7 ⁺ , CD45RA ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , IL-7Rα ⁺ , CD95 ⁺ , IL-2Rβ ⁺ , CXCR3 ⁺ and LFA-1 ⁺ ; b) are CD45RA ⁺ , CCR7 ⁺ , CD62L ⁺ and CD95 ⁺ ; c) are CD45RA ⁺ , CD45RO ⁺ , CCR7 ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , CD95 ⁺ and IL-2Rβ ⁺ ; d) are CD45RO ^- , CD45RA ⁺ , CCR7 ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , CD127 ⁺ and CD95 ⁺ ; e) are CD45RA ⁺ , CD44 ^+/- , CD62L ⁺ , CD127 ⁺ , IL-2Rβ ⁺ , CD28 ⁺ , CD43 ^- , KLRG1 ^- , Perforin ^- and GranzymB ^- ; f) express high levels of CCR7, CD62L, CD27 and CD28, intermediate levels of CD95 and IL-2Rβ, low levels of CD45RA, and do not express CD45RO or KLRG-1; or g) express high levels of CD62L, low levels of CD44 and t-bet and are Sca-1 ⁺ ; and/or have intermediate IL-2 production capacity, low IFNγ production capacity, low cytotoxicity, and high self-renewal capacity.

In some embodiments, a subset of T cells in cultured T cells (e.g., a subset of T cells that is increased in cultured T cells compared to a reference composition or formulation) constitutes or contains memory T cells, such as long-lived T cells memory. In some embodiments, the memory T cells are central memory T cells (Tcm). In some embodiments, a subset of T cells have a phenotypic characteristic of CD45RA-, CD45RO ^low/+ , CCR7+, CD62L+, CD27+, CD28+, CD95+ CD122+ and/or KLGR1 ^low . In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, the memory T cells are stem central memory T cells (Tscm). In some embodiments, a subset of T cells have the phenotypic characteristic of CD45RA ^low/+ , CD45RO ^low/+ , CCR7+, CD62L+, CD27+, CD28+, CD95+, CD122+ and/or KLGR1-. In some embodiments, a subset of T cells have the phenotypic characteristic of CD45RA ^low/+ , CD45RO ^- , CCR7+, CD62L+, CD27+, CD28+, CD95+, CD122+ and/or KLGR1-. In some embodiments, a subset of T cells has a phenotypic characteristic of CD45RO ^- , CCR7 ⁺ , CD45RA ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , IL-7Rα ⁺ , CD95 ⁺ , IL-2Rβ ⁺ , CXCR3 ⁺ and/or LFA-1 ⁺ . In some embodiments, a subset of T cells has a phenotypic characteristic of CD45RA ⁺ , CCR7 ⁺ , CD62L ⁺ and/or CD95 ⁺ . In some embodiments, a subset of T cells has a phenotypic characteristic of CD45RA ⁺ , CD45RO ⁺ , CCR7 ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , CD95 ⁺ and/or IL-2Rβ ⁺ . In some embodiments, a subset of T cells has a phenotypic characteristic of CD45RO ^- , CD45RA ⁺ , CCR7 ⁺ , CD62L ⁺ , CD27 ⁺ , CD28 ⁺ , CD127 ⁺ and/or CD95 ⁺ . In some embodiments, a subset of T cells has a phenotypic characteristic of CD45RA ⁺ , CD44 ^+/- , CD62L ⁺ , CD127 ⁺ , IL-2Rβ ⁺ , CD28 ⁺ , CD43 ^- , KLRG1 ^- , Perforin ^- and/or Granzyme B ^- . In some embodiments, a subset of T cells express high levels of CCR7, CD62L, CD27 and/or CD28, intermediate levels of CD95 and/or IL-2Rβ, low levels of CD45RA, and/or do not express CD45RO and/or KLRG-1. In some embodiments, a subset of T cells express high levels of CD62L, low levels of CD44 and t-bet, and/or are Sca-1 ⁺ . In some embodiments, the subset of T cells has a phenotypic characteristic of intermediate IL-2 production, low IFNγ production, low cytotoxicity, and/or high self-renewal. In some embodiments, such a subset of T cells in cultured T cells is at least about 2-fold increased (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9 times, 10 times or more) compared to the number or percentage of a subset of T cells in a reference composition or T cell culture.

In some embodiments, a subset of T cells, such as any of the T cell subsets described above, is present at a greater percentage of the total T cells in the cultured T cells or a greater number of the total T cells in the cultured T cells by compared to a reference composition or T-cell preparation. In some embodiments, the percentage of the T cell subset in cultured T cells, as a percentage of total T cells or total cells in culture, is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more. In some embodiments, the percentage of a subset of T cells in cultured cells, such as any of the T cell subsets described above, is greater, such as at least 1.5 times greater, at least 2 times, at least 3 times times, at least 4 times, at least 5 times, or more than the corresponding percentage of a subset of cells in a T cell composition isolated or enriched directly from a human subject based on surface expression of one of the phenotype-containing markers, but without incubation or cultivation. In some embodiments, the total number, relative number, or normalized amount of a subset of T cells in cultured cells, e.g., any T cell subset described above, is greater, such as at least 1.5 times, at least 2 times , at least 3 times, at least 4 times, at least 5 times, or more than the number, relative amount, or normalized amount of a subset of T cells in a reference composition or T cell preparation, e.g., any of those described above a reference composition or T cell preparation, e.g., a T cell composition prior to incubation with a reversibly coupled agent (e.g., a multimerized agent, e.g., prior to incubation with a stimulatory agent reversibly bound to a streptavidin oligomeric mutein) according to any of the methods provided herein . In some embodiments, the number of T cells corresponding to the subset of T cells present in the T cell culture is at least or at least about 1x10 ⁶ cells, 2x10 ⁶ cells, 3x10 ⁶ cells, 4x10 ⁶ cells, 5×10 ⁶ cells or more.

In some embodiments, the T cell subset is CD62L+ and/or IL-7Rα+ (CD127+) and the percentage of the CD62L+ and/or IL-7Rα+ (CD127+) subset in cultured T cells as a percentage of total T- cells or the total number of cells in culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In some embodiments, the subset of T cells is CD45RA-, CD45RO ^low/+ , and/or KLRG1 ^low and the percentage of the subset of CD45RA-, CD45RO ^low/+ , and/or KLRG1 ^low in cultured T cells as a percentage of the total T cells or total cells in culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 % or more. In some embodiments, the subset of T cells is CD45RA ^low/+ , CD45RO ^low/+ and/or KLRG1 ^- and the percentage of the subset of CD45RA ^low/+ , CD45RO ^low/+ and/or KLRG1 ^- in cultured T cells as a percentage the value of the total number of T cells or the total number of cells in culture is at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In some embodiments, the subset of T cells constitutes or contains Tcm cells. In some embodiments, the percentage of the Tcm subset in cultured T cells as a percentage of total T cells or total cells in culture is at least 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In some embodiments, the subset of T cells constitutes or contains Tscm cells. In some embodiments, the percentage of the Tscm subset in cultured T cells as a percentage of total T cells or total cells in culture is at least 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95% or more.

In some embodiments, a subset of T cells, such as CD62L+ T cells, have or exhibit a) a low level of TCR rearrangement excision rings (TRECs); and/or b) express a proliferation marker (eg, Ki-67); and/or c) demonstrate the ability to proliferate in the presence of a stimulating agent; and/or d) demonstrate the ability to produce a cytokine selected from among IFN-gamma, TNF and IL-2 in the presence of a stimulatory agent; and/or e) are resistant to depletion in the absence of a stimulating agent; and/or f) capable of generating Tscm, Tcm, Tem and Teff cells; and/or g) have low cytotoxicity; and/or h) can generate the same or greater response after adoptive transfer of fewer cells than with Tcm or Tem cells. In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine (eg, IL-15 or IL-17), or is an agent that is capable of initiating a TCR/CD3 complex-associated signal in T cells. In some embodiments, the ability to produce a cytokine includes a low ability to produce IFNγ and/or an intermediate ability to produce IL-2.

In some embodiments, cultured T cells are provided, for example, obtained according to any of the methods presented herein, wherein the cultured T cells are created or obtained after incubation, as described herein, and, while the cultured T cells are different modified profile of functional activity compared to the reference composition or preparation of T-cells. In some embodiments, the cultured T cells, or a specific subset of T cells present in culture, exhibit an altered functional activity profile compared to a reference composition or formulation, or compared to a subset of T cells in a reference composition or formulation, e.g., functional activity, which is changed (for example, increased or reduced) by at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times. In some embodiments, the functional activity is selected from one or more of a) low excision ring levels in TCR rearrangement (TREC); and/or b) expression of a proliferation marker (eg Ki-67); and/or c) the ability to proliferate in the presence of a stimulating agent; and/or d) the ability to produce a cytokine selected from IFN-gamma, TNF and IL-2 in the presence of a stimulatory agent; and/or e) resistance to depletion in the absence of a stimulating agent; and/or f) the ability to generate Tscm, Tcm, Tem and Teff cells; and/or g) low cytotoxicity. In some embodiments, the stimulatory agent is an antigen, a homeostatic cytokine (eg, IL-15 or IL-17), or is an agent that is capable of initiating a TCR/CD3 complex-associated signal in T cells. In some embodiments, the ability to produce a cytokine includes a low ability to produce IFNγ and/or an intermediate ability to produce IL-2. In some embodiments, the subset of T cells contains memory T cells, such as long-lived memory T cells, in cultured T cells. In some embodiments, the memory T cells are Tscm cells.

In some embodiments, cultured T cells, or a particular subset of T cells present in culture, can produce the same or greater response after adoptively transferring fewer cells than can be provided by a reference composition or formulation, or by a subset of T cells in reference composition or preparation. In some embodiments, such a response is achieved with at least about 2-fold fewer cells (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10 times or more). In some embodiments, the response increases or becomes at least about 2-fold greater (e.g., any of at least about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more).

In some embodiments, the percentage of the subset of T cells in the cultured cells, e.g., any of the T cell subsets described above, is greater, such as at least 1.5-fold, at least 2-fold, at least 3 times, at least 4 times, at least 5 times or more than the corresponding subset of cells in the preparation of T cells that were incubated in the presence of a GSK-P inhibitor. In some embodiments, the cultured T cell composition does not contain a GSK-P inhibitor.

In some embodiments, the percentage of the subset of T cells in the cultured cells, e.g., any of the T cell subsets described above, is greater, such as at least 1.5-fold, at least 2-fold, at least 3 times, at least 4 times, at least 5 times or more than the corresponding subset of cells that were incubated in the presence of a recombinant homeostatic cytokine, optionally IL-7 or IL-15. In some embodiments, the cultured T cell composition does not contain recombinant (eg, exogenous) IL-7 cytokine or recombinant (eg, exogenous) IL-15 cytokine.

In some embodiments, a composition of cultured T cells is prepared or created according to any of the methods provided herein, in which a substance, such as a competitive agent, is added to the T cells to disrupt, for example, attenuate and/or stop signaling of stimulatory agents or agent. In some embodiments, the cultured T cell composition contains a substance such as a competitive agent, such as biotin or a biotin analog, such as D-biotin. In some embodiments, a substance, such as a competitive agent, such as biotin or a biotin analog, such as D-biotin, is present in an amount greater than at least 1.5 times, at least 2 times, at least 3 times , at least 4 times, at least 5 times, at least 10 times, at least 100 times, at least 1000 times or more than the amount of the substance in the reference composition or cultured T-cell preparation in which the substance was not added exogenously during incubation. In some embodiments, the amount of a substance, such as a competitive agent, e.g., biotin or a biotin analog, e.g., D-biotin, in the cultured T cell composition is from or about 10 μM to 100 μM, 100 μM to 1 mM, 100 µM to 500 µM or 10 µM to 100 µM.

IV. Genetic Engineering Methods for Cultured Cells, Antigen Receptors and Genetically Engineered Cells

In some embodiments, the cultured cells contain or are engineered to contain an engineered receptor, for example, an engineered antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Also provided are populations of such cells, compositions comprising such cells and/or enriched in such cells, for example enriched in or selected with certain cell types such as T cells or CD8+ or CD4+ cells. The compositions include pharmaceutical compositions and formulations for administration, for example for adoptive cell therapy. Also provided are therapeutic methods for administering cells and compositions to subjects, such as patients.

Thus, in some embodiments, the cultured cells contain one or more genetically engineered nucleic acids and thus express recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is performed by first stimulating the cultured cells, such as by combining it with a stimulus that elicits a response such as proliferation, survival, and/or activation, such as as measured by cytokine expression or marker activation, followed by transduction of activated cells and propagation in culture to a quantity sufficient for clinical use.

In some contexts, overexpression of a stimulatory factor (eg, lymphokine or cytokine) may be toxic to the subject. Thus, in some contexts, engineered cells contain gene segments that cause cells to be susceptible to negative selection in vivo, such as when administered in adoptive immunotherapy. For example, in some aspects, the cultured cells are engineered such that they can be eliminated by changing the in vivo state of the patient to whom they are administered. The negatively selected phenotype may result from the insertion of a gene that confers sensitivity to the administered agent, eg a compound. Negatively selected genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, I977), which confers sensitivity to ganciclovirus; cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al. Proc. Natl. Acad. Sci. USA. 89:33 (1992)). In some aspects, the cultured cells are further engineered to stimulate the expression of cytokines or other factors.

A. Nucleic acids encoding antigen receptors, such as chimeric antigen receptors

Methods, nucleic acids, compositions and kits for obtaining genetically engineered cells are presented. Genetic engineering typically involves introducing a nucleic acid encoding a recombinant or engineered component into a composition containing cultured cells, for example, by retroviral transduction, transfection, or transformation.

In some embodiments, the nucleic acids are heterologous, ie. Typically not present in a cell or a sample derived from a cell, such as a sample derived from another organism or cell, which, for example, is not normally found in the engineered cell and/or the organism from which the cell is derived. In some embodiments, the nucleic acids are not naturally occurring, for example, a nucleic acid not found in nature, including an acid containing chimeric combinations of nucleic acids encoding different domains from a variety of different cell types.

1. Chimeric antigen receptors (CAR)

cells typically express recombinant receptors such as antigen receptors, including functional non-TCR antigen receptors such as chimeric antigen receptors (CAR) and other antigen-binding receptors such as transgenic T cell receptors (TCR). There are also other chimeric receptors between the receptors.

Exemplary antigen receptors, including CARs, and methods for constructing and introducing such receptors into cells include the receptors and methods described, for example, in International Patent Application Publication Nos. 166321, WO 2013/071154, WO 2013/123061, публикациях заявок на выдачу патента США номера US 2002131960, US 2013287748, US 20130149337, патентах США №№: 6451995, 7446190, 8252592, 8339645, 8398282, 7446179, 6410319, 7070995, 7265209 , 7354762, 7446191, 8324353 and 8479118 and European patent application number EP 2537416 and/or the applications described in Sadelain et al. Cancer Disks. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al. Curr. Opin. Immunol. October 2012; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, antigen receptors include CAR, which is described in US patent no: 7446190, and the receptors described in international patent application publication no: WO/2014055668 A1. Examples of CARs include those disclosed in any of the above publications such as WO 2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No: 7446190, US Patent No: 8389282, Kochenderfer et al. 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al. Sci Transl Med. 2013 5(177). See also WO 2014031687, US 8339645, US 7446179, US 2013/0149337, US Patent No: 7446190, and US Patent No: 8389282. Chimeric receptors, such as CAR, typically contain an extracellular antigen binding domain, such as part of an antibody molecule, usually the variable region of the heavy (V _H ) chain and/or the variable region of the light (V _L ) chain of an antibody, for example, an scFv fragment of an antibody.

In some embodiments, the antigen secreted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells associated with a disease or condition, such as tumor or pathogenic cells, compared to normal or unisolated cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on engineered cells.

Receptor-releasing antigens, in some embodiments, include orphan tyrosine kinase receptor RORl, tEGFR, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, 0EPHa2, ErbB2, 3 or 4, FBP, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr , kappa light chain, Lewis Y, cell adhesion molecule L1, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligands, NY-ESO-1, MART-1, gp100, oncoembryonic antigen, ROR1, TAG72, VEGF-R2 , cancer embryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin-B2, CD123, c-Met, GD-2 and MAGE A3, CE7, Wilms tumor gene (WT -1), cyclin, eg cyclin A1 (CCNA1), and/or biotinylated molecules and/or molecules expressed by HIV, HCV, HBV or other pathogens.

In some embodiments of any of the methods provided herein, the target cells express a CAR that binds to an antigen associated with a disease and/or cancer. In specific embodiments of any of the methods provided herein, the antigen is an αvβ6 integrin (avb6 integrin), B-cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), cancer- testicular antigen, cancer testicular antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), cancer embryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19 , CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein ( tEGFR), epidermal growth factor receptor type III (EGFR vIII) mutation, epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin-B2, ephrin A2 receptor (EPHa2), estrogen receptor, Fc receptor -like protein 5 (FCRL5; also known as Fc receptor homologue 5 or FCRH5), fetal acetylcholine receptor (embryonic AchR), folate binding protein (FBP), folic acid receptor alpha, embryonic acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), G-protein coupled receptor 5D (GPCR5D), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, high molecular weight human melanoma-associated antigen (HMW-MAA), hepatitis virus surface antigen B, human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2 (HLA-A2), IL-22 alpha receptor (IL-22Ra), IL-13 alpha receptor 2 (IL-13Ra2), receptor containing kinase insertion domain (kdr), kappa light chain, cell adhesion molecule L1 (L1CAM), CE7 epitope L1-CAM, leucine-rich repeat member A family 8 (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegavirus (CMV), mucin 1 (MUC1), MUC16, ligand natural killer group D 2 (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncoembryonic antigen, preferentially expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen gland (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor gene (WT-1), pathogen-specific or universal label-associated antigen and/or biotinylated molecules and/or molecules expressed by HIV, HCV , HBV or other pathogens.

In some embodiments, the CAR binds a pathogen-specific antigen. In some embodiments, the CAR is specific for viral antigens (such as HIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

In some embodiments, the recombinant receptor antibody portion, e.g., CAR, further comprises at least a portion of an immunoglobulin constant region, such as a hinge region, e.g., an IgG4 hinge region and/or CH1/CL and/or an Fc region. In some embodiments, the implementation of the constant region or part refers to a human IgG, such as IgG4 or IgG1. In some aspects, a portion of the constant region serves as a spacer region between the antigen recognition component, eg, scFv, and the transmembrane domain. The spacer may be of a length that provides for an increased cell response after antigen binding compared to no spacer. Exemplary spacers, such as hinge regions, include those described in International Patent Application Publication Number WO2014031687. In some examples, the spacer is or approximately 12 amino acids long, or no more than 12 amino acids long. Exemplary spacers include spacers having at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids , about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, the spacer region is about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include only an IgG4 hinge, an IgG4 hinge associated with the CH2 and CH3 domains, or an IgG4 hinge associated with the CH3 domain.

This antigen recognition domain is typically associated with one or more intracellular signaling components, such as signaling components that mimic activation via an antigen-receptor complex, such as a TCR complex in the case of a CAR, and/or a signal via another cell surface receptor. Thus, in some embodiments, an antigen-binding component (eg, an antibody) is associated with one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain is used that is naturally associated with one of the domains in the receptor, such as CAR. In some instances, the transmembrane domain is chosen or modified by amino acid substitution to avoid binding of such domains to transmembrane domains, proteins of the same or different surface membranes, to minimize interactions with other elements of the receptor complex.

The transmembrane domain, in some embodiments, is delivered from either a natural or synthetic source. When the source is natural, the domain is in some aspects delivered from any membrane-bound or transmembrane protein. Transmembrane regions include regions derived from (i.e., contain at least the transmembrane region(s)) T cell receptor alpha, beta or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD 16 , CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154. Alternatively, the transmembrane domain is synthetic in some embodiments. In some aspects, the synthetic transmembrane domain contains predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be located at each end of the synthetic transmembrane domain. In some embodiments, communication is via linkers, spacers, and/or transmembrane domain(s).

Among the intracellular signaling domains are those that mimic or approximate a signal via a natural antigen receptor, a signal via such a receptor in combination with a costimulatory receptor, and/or a signal via a costimulatory receptor alone. In some embodiments, a short oligo or polypeptide linker, e.g., a linker between 2 and 10 amino acids in length, such as a linker containing glycines and serines, e.g., a glycine-serine doublet, is present and forms a link between the transmembrane domain and the CAR cytoplasmic signaling domain.

A receptor, such as a CAR, typically contains at least one intracellular signaling component or components. In some embodiments, the receptor contains an intracellular component of the TCR complex, such as the CD3 TCR chain, which mediates T cell activation and cytotoxicity, such as the CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is associated with one or more signaling modules of the cell. In some embodiments, cell signaling modules include a CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the implementation of the receptor, for example, CAR, further comprises part of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25 or CD16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

In some embodiments, upon ligation of a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of an immune cell, such as a T cell engineered to express the CAR. For example, in some contexts, CAR induces T cell function, such as cytotoxic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or a co-stimulatory molecule is used instead of an intact immunostimulatory chain, for example if it transduces an effector function signal. In some embodiments, the intracellular signaling domain or domains comprise cytoplasmic T cell receptor (TCR) sequences, and in some aspects also co-receptor sequences, which in a natural context act in conjunction with such receptors to initiate signal transduction following antigen-receptor interaction.

In the context of the natural TCR, full activation usually requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, the CAR also contains a component to generate a secondary or co-stimulatory signal to stimulate full activation. In other embodiments, the implementation of the CAR does not contain a component to create a costimulatory signal. In some aspects, the additional CAR is expressed in the same cell and provides a component to create a secondary or co-stimulatory signal.

T cell activation has in some aspects been described as being mediated by two classes of cytoplasmic signal sequences: those that initiate antigen-dependent primary activation via TCRs (primary cytoplasmic signal sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal ( secondary cytoplasmic signal sequences). In some aspects, the CAR contains one or both of these signaling components.

In some aspects, the CAR contains a primary cytoplasmic signal sequence that regulates the primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, which are known as immunoreceptor tyrosine activating motifs or ITAMs. Examples of ITAMs containing primary cytoplasmic signal sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule(s) in the CAR comprises (comprises) a cytoplasmic signaling domain, a portion or sequence thereof derived from CD3 zeta.

In some embodiments, the CAR contains a signaling domain and/or a transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, OX40, DaP10, and ICOS. In some aspects, the same CAR contains both activating and costimulatory components.

In some embodiments, the activating domain is contained in one CAR while the co-stimulatory component is provided by other CARs recognizing a different antigen. In some embodiments, CARs include activating or stimulating CARs co-stimulating CARs, both of which are expressed on the same cell (see WO2014/055668). In some aspects, the cells contain one or more stimulatory or activating CARs and/or costimulatory CARs. In some embodiments, cells further comprise inhibitory CARs (iCARs, see Fedorov et al. Sci. Transl. Medicine, 5(215) (December, 2013), such as antigen-recognizing CARs that are not antigen-related and/or specific to a disease or condition whereby an activating signal delivered through a disease-targeted CAR is attenuated or inhibited by binding the inhibitory CAR to its ligand, for example, to reduce unwanted outcomes.

In some embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain associated with an intracellular CD3 domain (eg, CD3 zeta). In some embodiments, the intracellular signaling domain comprises CD28 and CD137 chimeric co-stimulatory domains (4-1BB, TNFRSF9) associated with the CD3 zeta intracellular domain.

In some embodiments, the implementation of the CAR encompasses one or more, for example, two or more costimulatory domains and an activating domain, for example, the primary activating domain, in the cytoplasmic part. Exemplary CARs contain the intracellular components CD3-zeta, CD28, and 4-1BB.

In some embodiments, the CAR or other antigen receptor further comprises a marker, such as a cell surface marker, which can be used to confirm transduction or engineer the cell to express a receptor, such as a truncated version of the cell surface receptor, such as truncated EGFR (tEGFR). In some aspects, the marker contains all or part (eg, a truncated form) of CD34, NGFR, or epidermal growth factor receptor (eg, tEGFR). In some embodiments, a nucleic acid encoding a marker is operably linked to a polynucleotide encoding a linker sequence, such as a cleavable linker sequence, for example, T2A. See WO2014031687.

In some embodiments, the marker is a molecule, such as a cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.

In some embodiments, the molecule is not its own molecule, e.g., not its protein, i.e. a protein that is not recognized as "self" by the host's immune system, into which cells will be adoptively transferred.

In some embodiments, the implementation of the marker does not have a therapeutic function and/or does not create any effect, except for use as a marker for genetic engineering, for example, to select successfully engineered cells. In other embodiments, the marker may be a therapeutic molecule or a molecule otherwise having a specific desired effect, such as a ligand for a cell encountered in vivo, such as a co-stimulatory molecule or an immune checkpoint molecule to enhance and/or attenuate cell responses in adoptive transfer, and collision with a ligand.

In some cases, CARs are referred to as first, second and/or third generation CARs. In some aspects, a first generation CAR is a CAR that provides only an induced CD3 chain signal upon antigen binding; in some aspects, a second generation CAR is a CAR that provides such a signal and a costimulatory signal, for example, a CAR containing an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is a CAR that contains multiple costimulatory domains of different costimulatory receptors.

In some embodiments, the implementation of the chimeric antigen receptor contains an extracellular part containing the antibody or antibody fragment. In some aspects, the chimeric antigen receptor contains an extracellular portion containing the antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment contains scFv and the intracellular domain contains ITAM. In some aspects, the intracellular signaling domain comprises the CD3-zeta (CD3ζ) chain zeta chain signaling domain. In some embodiments, the implementation of the chimeric antigen receptor contains a transmembrane domain that binds an extracellular domain and an intracellular signaling domain. In some aspects, the transmembrane domain contains the transmembrane portion of CD28. In some embodiments, the implementation of the chimeric antigen receptor contains the intracellular domain of the T-cell co-stimulatory molecule. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including provided receptors and other polypeptides, such as linkers or peptides, may contain amino acid residues, including natural and/or non-natural amino acid residues. the terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications relative to the native or natural sequence, provided that the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as mutations in hosts that produce proteins, or errors due to PCR amplification.

In some embodiments, the implementation of the receptor, for example, CAR, expressed by cells in a sequential dose, as a receptor contains at least one immunoreactive epitope, for example, CAR, expressed by cells of the first dose. In some aspects, the receptor, eg, CAR, expressed by the cells administered at the sequential dose is identical to the receptor, eg, CAR, expressed by the first dose, or substantially identical to the receptor, eg, CAR, expressed by the cells administered at the first dose.

Recombinant receptors, such as CARs, expressed by cells administered at different doses to a subject, typically recognize or bind specifically to a molecule that is expressed on cells, associated with cells, and/or is specific to cells associated with the disease or condition being treated. When specifically bound to a molecule, such as an antigen, the receptor typically delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby stimulating an immune response that targets the disease or condition. For example, in some embodiments, the cells at the first dose express a CAR that specifically binds to an antigen expressed by, or associated with, the disease or condition, cell or tissue.

2.TCR

In some embodiments, the genetically engineered antigen receptors include recombinant T cell receptors (TCRs) and/or TCRs cloned from naturally occurring T cells. In some embodiments, a clone of high affinity T cells for a target antigen (eg, a cancer antigen) is identified, isolated from a patient, and injected into the cells. In some embodiments, a TCR clone for a target antigen is generated from transgenic mice engineered with human immune system genes (eg, human leukocyte antigen or HLA system). See, for example, tumor antigens (See, for example, Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808. In some embodiments, phage display is used to isolate the TCR against the target antigen (see e.g. Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.

In some embodiments, after a T cell clone has been obtained, the alpha and beta TCR chains are isolated and cloned into a gene expression vector. In some embodiments, the alpha and beta TCR genes are linked via a picornavirus 2A ribosome slip-through peptide such that both strands are co-expressed. In some embodiments, TCR genetic transfer is performed via retroviral or lentiviral vectors or via transposons (see, for example, Baum et al. (2006) Molecular Therapy: Journal of American Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010) Molecular Therapy: Journal of American Society of Gene Therapy 18:1748-1757 Hackett et al (2010) Molecular Therapy: Journal of American Society of Gene Therapy 18:674-683

3. Multipurpose impact

In some embodiments, cells and methods include multipurpose strategies, such as the expression of two or more genetically engineered receptors on a cell, each of which recognizes the same or a different antigen, and typically each contains a different intracellular signaling component. Such multi-target strategies are described, for example, in International Patent Publication No: WO 2014055668 A1 (describing combinations of activating and co-stimulatory CARs, e.g. highlighting two different antigens present separately not on the target, e.g. on normal cells, but present together only on cells associated with the disease or condition being treated) and Fedorov et al. sci. Transl. Medicine, 5(215) (December, 2013) (describes cells expressing activating and inhibitory CAR, such as cells in which activating CAR binds to a single antigen expressed both on normal or non-diseased cells and on cells associated with the disease or condition to be treated, and the inhibitory CAR binds to another antigen expressed only on normal cells or cells that do not need to be treated).

For example, in some embodiments, the cells contain a receptor that expresses a first genetically engineered antigen receptor (e.g., CAR or TCR) that is capable of inducing an activating signal to the cell, typically upon specific binding to an antigen recognized by the first receptor, e.g., the first antigen. In some embodiments, the cell further comprises a second genetically engineered antigen receptor (e.g., CAR or TCR), e.g., a chimeric costimulatory receptor, that is capable of inducing a costimulatory signal to an immune cell, typically by specific binding to a second antigen recognized by the second receptor. In some embodiments, the first antigen and the second antigen are the same. In some embodiments, the first antigen and the second antigen are different.

In some embodiments, the implementation of the first and/or second genetically engineered antigen receptor (eg, CAR or TCR) is capable of inducing an activating signal to the cell. In some embodiments, the implementation of the receptor contains an intracellular signaling component containing ITAM or ITAM-like motifs. In some embodiments, activation induced by the first receptor includes signal transduction or a change in protein expression in a cell that results in the initiation of an immune response such as ITAM phosphorylation and/or initiation of an ITAM-mediated signal transduction cascade, formation of an immune synapse, and/or clustering of molecules around an associated receptor (e.g., CD4 or CD8, etc.), activation of one or more transcription factors such as NF-κB and/or AP-1, and/or induction of gene expression of factors such as cytokines, proliferation, and /or survival.

In some embodiments, the first and/or second receptor comprises intracellular co-stimulatory receptor signaling domains such as CD28, CD137 (4-1 BB), OX40, and/or ICOS. In some embodiments, the implementation of the first and second receptors contain different intracellular signaling domains of co-stimulatory receptors. In one embodiment, the first receptor contains the CD28 costimulatory signaling region and the second receptor contains the 4-1BB costimulatory signaling region, or vice versa.

In some embodiments, the first and/or second receptor contains both an intracellular signaling domain containing ITAM or ITAM-like motifs and an intracellular signaling domain of a costimulatory receptor.

In some embodiments, the first receptor contains an intracellular signaling domain containing ITAM or ITAM-like motifs, and the second receptor contains an intracellular signaling domain of a costimulatory receptor. A costimulatory signal, in combination with an activating signal induced in the same cell, is a signal that results in an immune response, such as a strong and delayed immune response, such as increased gene expression, secretion of cytokines and other factors, and T-cell mediated effector functions. such as killing cells.

In some embodiments, neither ligation of the first receptor alone nor ligation of the second receptor alone elicits a strong immune response. In some aspects, if only one receptor is ligated, the cell becomes tolerized or unresponsive to the antigen or inhibited and/or cannot be caused to proliferate or secrete factors or perform effector functions. In some such embodiments, however, when a plurality of receptors are ligated, for example, when a cell expressing the first and second antigens meets, the desired response is achieved, such as complete immune activation or stimulation, for example, as indicated by the secretion of one or more cytokines, proliferation, persistence and/or performing an immune effector function such as cytotoxic killing of target cells.

In some embodiments, the two receptors induce, respectively, an activating and inhibitory signal to a cell such that binding by one of the receptors to its antigen activates the cell or elicits a response, but binding by the second inhibitory receptor to its antigen elicits a signal that suppresses or attenuates that response. Examples are combinations of activating CARs and inhibitory CARs or iCARs. For example, a strategy can be used in which an activating CAR binds an antigen that is expressed in the disease or condition but is also expressed on normal cells, and an inhibitory receptor binds to a single antigen that is expressed on normal cells but not on cells associated with disease or condition.

In some embodiments, a multi-target strategy is used when an antigen associated with a particular disease or condition is expressed on a non-diseased cell and/or expressed on the engineered cell itself, either transiently (e.g., upon stimulation due to genetic engineering) or permanently. In such cases, specificity, selectivity and/or efficacy can be improved by the necessary ligation of two separate and individually specific antigen receptors.

In some embodiments, a plurality of antigens, such as the first and second antigens, are expressed on a cell, tissue, or isolated disease or condition, such as a cancer cell. In some aspects, the cell, tissue, disease, or condition is multiple myeloma or a multiple myeloma cell. In some embodiments, one or more of the plurality of antigens is typically also expressed on a cell that does not need to be isolated by cell therapy, such as a normal or non-diseased cell or tissue, and/or on the engineered cells themselves. In such embodiments, specificity and/or efficacy is achieved by requiring ligation of multiple receptors to achieve a cell response.

B. Vectors and methods of genetic engineering

Various methods of introducing genetically engineered components, for example, antigen receptors, such as CAR or TCR, are well known and can be used with the presented methods and compositions. Illustrative methods include methods for transferring receptor-encoding nucleic acids, including via viral, eg, retroviral or lentiviral, transduction, transposons, and electroporation.

In some embodiments, recombinant nucleic acids are transferred into cultured cells using particles of recombinant infectious viruses, such as, for example, vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors such as retroviral gamma vectors (see, for example, Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/ gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al. Trends Biotechnol. 2011 November 29( 11): 550-557.

In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, e.g., a retroviral vector derived from Moloney mouse leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen necrosis virus (SFFV), or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include retroviruses derived from any source of avian or mammalian cells. Retroviruses are generally amphotropic, in the sense that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the gag, pol and/or env retroviral sequences. a number of exemplary retroviral systems have been described (e.g., US Pat. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

methods of lentiviral transduction are known. Illustrative methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.

In some embodiments, recombinant nucleic acids are transferred into T cells by electroporation (see, for example, Chicaybam et al (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, for example, Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74 and Huang et al (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (eg, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; microparticle bombardment facilitated by tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transferring nucleic acids encoding recombinant products are those described in, for example, International Patent Application Publication No: WO2014055668 and US Patent No. 7446190.

In some embodiments, cells, such as T cells, may be transfected either during or after expansion, such as with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection to introduce the desired receptor gene can be done with any suitable retroviral vector, for example. the genetically modified population of cells can then be released from the original stimulus (eg, CD3/CD28 stimulus) and then stimulated with a second type of stimulus, eg, via a de novo injected receptor). This second type of stimulus may include a peptide/MHC antigenic stimulus, a cognate (cross-linking) ligand of a genetically introduced receptor (eg, a naturally occurring CAR ligand), or any ligand (such as an antibody) that binds directly within the scaffold of the new receptor (eg, through recognition constant regions within the receptor). See, for example, Cheadle et al, "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907:645-66 or Barrett et al. Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65:333-347 (2014).

Among additional nucleic acids, for example, genes for introduction are genes that improve the efficacy of therapy, for example, by promoting the viability and/or function of the transferred cells; genes for providing a genetic marker for selection and/or evaluation of cells, for example, for evaluation of survival or localization in vivo; genes to improve safety, for example, making the cell susceptible to negative selection in vivo, as described in Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al. Human Gene Therapy 3:319-338 (1992); see also PCT/US91/08442 and PCT/US94/05601 Lupton et al. describing the use of bifunctional selective fusion genes resulting from the fusion of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al. US Patent No. 6040177 columns 14-17.

In some cases, you can use a vector that does not require activation of cells, such as T cells. In some such cases, cells can be selected and/or transduced prior to activation. Thus, cells can be constructed before or after cell culture as described herein, and in some cases at the same time or during at least part of the culture. In some embodiments, the cells to be constructed are cultured cells, or in some cases the cells can be transduced prior to culture, as described herein.

C. Cell Transduction Methods

In some embodiments, methods for transferring a viral vector into a cell are performed using any provided oligomeric protein reagent (eg, streptavidin or streptavidin mutein). In some embodiments, the oligomeric protein reagent used according to the provided transduction methods is a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents. In some embodiments, the cells are intended for use in cell therapy, such as primary cells obtained for autologous or allogeneic transfer, such as in adoptive cell therapy. In some embodiments, the reagent may also be used in methods to facilitate one or more other processing steps associated with obtaining an engineered cell composition, for example, one or more of cell selection or modulation, activation and/or stimulation. The methods may include additional steps for processing cells, for example, washing, isolating, separating, preparing cells, or other steps associated with obtaining a cell composition.

In some embodiments, the present methods are used to introduce viral vector particles, such as retroviral vector particles, into cells, such as immune cells, including T cells. In some embodiments, the viral vector particles have a genome that contains a nucleic acid encoding an antigen receptor, such as a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR). Therefore, in some embodiments, the present methods can be used to express in immune cells, such as T cells, a genetically engineered antigen receptor, such as a transgenic TCR or CAR. Also provided are cells transduced with such particles, and methods and compositions containing such cells, and methods of using them.

In some embodiments, retroviral vector particles and methods include features that result in increased transduction of immune cells and/or certain populations and/or subpopulations thereof that are desirable for use in adoptive immunotherapy. In some embodiments of the provided transduction methods and viral vector particles, cells, e.g., T cells, in the population to be transduced are not stimulated or do not need to be stimulated and/or activated before and/or in combination with contacting or incubating the cells with provided retroviral vector particles.

A. Cell Incubation with Virus Vector Particles

In some embodiments, the methods provided include methods of transducing cells by contacting, e.g., incubation, a cell composition comprising a plurality of cells (hereinafter also referred to as "input composition") with (1) an oligomeric protein (e.g., streptavidin mutein) reagent such as a multimerization reagent and/or an oligomeric particulate reagent associated with one or more agents, and (2) viral particles. In some embodiments, the method includes mixing the cells with the reagent and with the viral particles simultaneously or sequentially. In some embodiments, the method includes pre-mixing the virus particles and the reagent together and then contacting the cell composition with the mixture of virus particles associated with the reagent. In some embodiments, contacting occurs for 30 minutes to 72 hours, such as 30 minutes to 48 hours, 30 minutes to 24 hours, or 1 hour to 24 hours, such as at least or approximately at least within 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.

In some embodiments of any of the methods provided herein, (i) the incubation comprises mixing the target cells with the reagent and/or mixing the target cells with the viral particles, sequentially, in any order, optionally, where the mixing in (a) and mixing in (b) is carried out for a period of not more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours and/or mixing in (a) is carried out for no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 hours apart from mixing in (b); (ii) the incubation includes mixing the target cells, the reagent and the viral particles, said mixing being performed simultaneously or substantially simultaneously; (iii) incubation includes mixing a composition that contains target cells and viral particles and does not contain a reagent, optionally, while: not more than 5%, 10%, 20%, 30% or 40% of target cells in a composition containing cells -targets and reagent, are activated cells, express a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; including intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha, and/or proliferating; and / or mixing is carried out for no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 hours after mixing the target cells and viral particles in the composition; (iv) incubation includes mixing a composition that contains target cells and a reagent and does not contain viral particles with viral particles, optionally, with no more than 5%, 10%, 20%, 30% or 40% of the target cells in the composition containing target cells and reagent are activated cells expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB; including intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha, and/or proliferating; and / or mixing is carried out for no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 hours after mixing the target cells and viral particles in the composition; and/or (v) incubation involves mixing a composition containing viral particles and a reagent with a composition that contains target cells and not viral particles and/or no reagent, optionally, wherein: not more than 5%, 10%, 20 %, 30% or 40% of the target cells in the composition containing the target cells and the reagent are activated cells expressing a surface marker selected from the group consisting of HLA-DR, CD25, CD69, CD71, CD40L and 4-1BB ; including intracellular expression of a cytokine selected from the group consisting of IL-2, IFN-gamma, TNF-alpha, and/or proliferating.

In some embodiments, the implementation of the incubation includes mixing the cells with the reagent and with the viral particles, simultaneously or sequentially, in any order. In some embodiments, during at least part of the incubation, the reagent and the viral particles are both in the presence of or in contact with the cell.

In some embodiments, the methods provided include (a) contacting viral particles with an oligomeric protein reagent, thereby obtaining a composition comprising viral particles and a reagent, wherein the virus particles are optionally associated with a reagent; and (b) incubating the composition in (a) with a plurality of cells, including target cells, wherein the method produces an output composition containing one or more cells transduced with viral particles.

In some embodiments, the present methods include mixing a composition containing viral particles and an oligomeric protein reagent with a plurality of cells, including target cells, wherein the method produces an output composition containing one or more cells transduced with viral particles.

In some embodiments, the present methods comprise (a) contacting the viral particles with an oligomeric protein reagent containing streptavidin or a mutein or a biologically active fragment of any of the foregoing and/or a plurality of subunits of any of the foregoing, thereby obtaining a composition comprising the viral particles and a reagent, wherein the viral particles are optionally associated with the reagent; and (b) incubating the composition in (a) with a plurality of cells, wherein the method produces an output composition containing one or more cells transduced with viral particles.

In some embodiments, the methods provided include mixing a composition containing viral particles and a protein reagent with a plurality of cells, including target cells, wherein: the protein reagent contains streptavidin, avidin, avidin streptanalog or mutein, an avidin analog, mutein, or a biologically active fragment of any of the above and/or a plurality of subunits of any of the above; and in the method, an output composition is obtained containing one or more cells transduced with viral particles. In some embodiments, the reagent and/or each of the monomeric units and/or each of the multimeric units has a net positive charge or a net positive charge.

In some embodiments, the methods provided include incubating a plurality of cells, including target cells, with: 1) an oligomeric protein reagent containing a plurality of binding sites capable of reversibly binding to a binding agent, wherein one or more binding sites are reversibly associated with a binding agent; and 2) viral particles, wherein at least part of the incubation in (1) occurs simultaneously with (2), and, in this process, an output composition is obtained containing one or more cells transduced with viral particles.

In some embodiments, the methods provided include (1) contacting (a) a composition containing one or more viral particles, and (b) a binding agent, which is a virus-binding agent that (i) is capable of specifically binding to a molecule on a surface viral particles and ii) reversibly linked to a reagent containing a plurality of binding sites capable of reversibly binding to a virus-binding agent; and (2) incubating at least a plurality of cells, including target cells, in the presence of one or more viral particles, wherein the contacting in (1) and the incubation in (2) are performed simultaneously or sequentially, in any order, wherein the method creates an output composition containing a plurality of cells transduced with viral particles.

In some embodiments of any of the methods provided herein, contacting in (1) and incubation in (2) is carried out for a period of no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours or 72 hours, and/or mixing in (a) is carried out for no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours or 48 hours apart from the incubation in (b). In some embodiments, the viral vector particles comprise a genome encoding a recombinant antigen receptor, optionally a chimeric antigen receptor.

The composition that contains the viral vector particles and the cells during the transduction step may further contain one or more additional agents, such as agents to promote transduction efficiency, such as polycations, including protamine (for example, protamine sulfate), hexadimethrin bromide (POLYBRENE®, Abbott Laboratories Corp), and CH-296 (RETRONECTIN®, Clontech). In some embodiments, the implementation of the polycation may be present in the input composition with a final concentration of from 1 µg/ml to 100 µg/ml, for example, from 5 µg/ml to 50 µg/ml. The composition may also contain media, including cell culture media, including media designed for culture of the cell type to be treated, such as media for hematopoietic stem cells, for example, serum-free media.

In some embodiments, the cell concentration of the input composition is from or about 1.0 x 10 ⁵ cells/mL to 1.0 x 10 ⁸ cells/mL, such as at least or about at least or about 1.0 x 10 ⁵ cells/ml, 5x10 ⁵ cells/ml, 1x10 ⁶ cells/ml, 5x10 ⁶ cells/ml, 1x10 ⁷ cells/ml, 5x10 ⁷ cells/ml or 1x10 ⁸ cells /ml

In some embodiments, the viral particles are presented with some ratio of copies of the viral vector particles or infectious units (IUs) thereof to the total number of cells (IU/cell) in the input composition or the total number of cells to be transduced. For example, in some embodiments, the viral particles are present during contact at or about or at or at least or about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 or 60 IU of viral vector particles per cell.

In some embodiments, the viral vector particle titer is between or about 1x10 ⁶ IU/ml and 1x10 ⁸ IU/ml, e.g., between or about 5x10 ⁶ IU/ml and 5x10 ⁷ IU/ml eg at least 6×10 ⁶ IU/ml, 7×10 ⁶ IU/ml, 8×10 ⁶ IU/ml, 9×10 ⁶ IU/ml, 1×10 ⁷ IU/ml, 2×10 ⁷ IU/ml, 3×10 ⁷ IU/ml, 4×10 ⁷ IU/ml or 5x10 ⁷ IU/ml.

In some embodiments, transduction can be achieved at a MOI of less than 100, eg typically less than 60, 50, 40, 30, 20, 10, 5, or less.

In some embodiments, contacting is carried out in solution, for example, using a soluble oligomeric protein reagent (eg, streptavidin mutein) or a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents. In some embodiments, cells, oligomeric reagent, and viral particles are brought into contact in a volume of from or about 0.5 ml to 500 ml, e.g., from or from about 0.5 ml to 200 ml, from 0.5 ml to 100 ml , 0.5 ml to 50 ml, 0.5 ml to 10 ml, 0.5 ml to 5 ml, 5 ml to 500 ml, 5 ml to 200 ml, 5 ml to 100 ml, from 5 ml to 50 ml, 5 ml to 10 ml, 10 ml to 500 ml, 10 ml to 200 ml, 10 ml to 100 ml, 10 ml to 50 ml, 50 ml to 500 ml, 50 ml ml to 200 ml, 50 ml to 100 ml, 100 ml to 500 ml, 100 ml to 200 ml or 200 ml to 500 ml.

In some embodiments, when contacting in solution, e.g., using a soluble oligomeric protein reagent (e.g., streptavidin mutein), a contact can be made in which at least part of the contact is carried out using centrifugation, e.g., spinoculation (e.g., centrifuge inoculation). In some embodiments, the composition containing the cells, virus particles, and reagent can be rotated, typically at a relatively low force or speed, such as a speed lower than the speed used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (eg, at or about or at least 600 rpm, 1000 rpm, or 1500 rpm, or 1700 rpm). In some embodiments, the rotation is accelerated, for example, a relative centrifugal acceleration from or from about 100 g to 3200 g (for example, at or about at or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), when measured, for example, on the inner or outer wall of a chamber or cavity. the term "relative centrifugal acceleration" or RCF is usually understood as the effective acceleration applied to an object or substance (such as a cell, sample or granule and/or a point in a chamber or other rotating container), relative to the gravity of the earth, at a particular point in space compared to the axis of rotation. the value can be determined using well-known formulas, taking into account the force of gravity, the speed of rotation and the radius of rotation (distance from the axis of rotation and the object, substance or particle for which the RCF is measured).

In some embodiments, the oligomeric reagent, such as the multimerization reagent and/or the particulate oligomeric reagent, associated with one or more agents is not associated with a carrier, eg, not associated with a solid surface or stationary phase.

In some embodiments, an oligomeric reagent, such as a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, is immobilized on a carrier, such as a solid surface or stationary phase. In some embodiments, contacting is carried out in a stationary phase, for example, using a chromatography matrix on which a protein (streptavidin mutein) is immobilized, such as an oligomeric protein reagent (eg, streptavidin mutein). This document describes examples of such formats for use in conjunction with the presented methods. Thus, in some embodiments, the implementation of the transduction in the column can be performed according to the presented methods.

In some embodiments, the implementation of the input composition, which is introduced into contact with cells, contains activated cells. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90% or more of the cells, e.g., T cells, in the input composition are activated, e.g., in some cases surface positive for one or more of HLA-DR, CD25, CD69, CD71, CD40L and/or 4-1BB. In some embodiments, cells are activated with an activating agent, eg, in the presence of an anti-CD3/anti-CD28 antibody, prior to initiation of contact, eg, prior to initiation of transduction. Methods for propagating populations of T cells in vitro in the absence of exogenous growth factors or at low levels of exogenous growth factors are known in the art (see, for example, US Pat. No. 6,352,694 B1 and EP 0 700 430 B1). In general, such methods involve solid phase surfaces greater than 1 μm onto which various binding agents (eg, anti-CD3 and/or anti-CD28) are immobilized. For example, Dynabeads® CD3/CD28 (Invitrogen) are commercially available T cell expansion reagents that are homogeneous, 4.5 µm superparamagnetic, sterile, non-pyrogenic polystyrene beads coated with a mixture of affinity purified monoclonal antibodies against CD3 cell surface molecules. and CD28 on human T cells. In some embodiments, the implementation of the activating agent, for example, against CD3 and/or against CD28, can be immobilized on beads, such as magnetic beads.

In some embodiments, cell activation is also performed in the presence of IL-2 (eg, from or from about 50 IU/ml to 200 IU/ml, for example, or about 100 IU/ml). In some embodiments, activation is from or about 1 hour to 96 hours, 1 hour to 72 hours, 1 hour to 48 hours, 4 hours to 36 hours, 8 hours to 30 hours, or 12 hours to 24 hours. , for example, at least or about at least 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, or 72 hours. In some embodiments, the activation is carried out at a temperature of greater than or about greater than 25ºC, e.g., typically greater than or about greater than 32ºC, 35ºC, or 37ºC, such as at or about 37ºC ± 2ºC, such as at or about 37ºC.

In some embodiments, the cells are not activated with an activating agent, eg, in the presence of an anti-CD3/anti-CD28 antibody, prior to initiation of contact, eg, prior to initiation of transduction. In some embodiments, the implementation of the input composition, which is entered into contact with the cells, contains a lot of resting cells. In some embodiments, at least 40%, 50%, 60%, 70%, 80%, 90% or more of the T cells in a population are resting T cells, such as T cells that lack the T-activation marker. cells, for example, a surface marker or an intracellular cytokine or other marker, and/or T cells that are at the G ₀ or G ₀ G _1a stage of the cell cycle.

In certain aspects, the present methods allow transduction in T cells without the need for activation prior to contact and/or incubation with an oligomeric protein reagent, such as a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents. In some embodiments, the methods include transducing a population of T cells, which include quiescent or naïve T cells, with a viral vector in the presence of an oligomeric protein reagent (e.g., streptavidin) according to the present methods, without first activating and/or stimulating T- cells, i.e. before transduction. In some such embodiments, the present methods can be used to produce immune cells, such as T cells, for adoptive therapy that do not include the step of activating and/or stimulating T cells.

In some embodiments, the oligomeric protein (eg, streptavidin mutein) is unprotected.

In some embodiments, the oligomeric protein (e.g., streptavidin mutein) is a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents that has one or more binding agents associated with it that is capable of binding to a molecule on the surface. target cells (eg, T cells) or, in some cases, on the surface of the viral particles in the composition. In some embodiments, the binding agent is reversibly linked to a reagent containing a plurality of binding sites capable of reversibly binding to the agent. In some embodiments, the implementation of the incubation is carried out under conditions in which the agent binds, for example, specifically binds, to a molecule on a cell or viral particle. In some described embodiments, the oligomeric reagent has an agent or agents reversibly immobilized on it (associated with it) (for example, the first or second or third, etc.), which may contain a receptor-binding, for example, a stimulating agent or additional agents, a selective agent or virus-binding agents that can be used to select, stimulate, expand and/or differentiate cells or modulate cell transduction.

In some cases, for certain receptor-binding agents (eg, stimulant agents or additional agents), such binding may induce or modulate a signal in target cells (eg, T cells) in the compositions, such as the described primary signal or an additional signal. In some embodiments, binding of an agent to a molecule results in one or more of stimulation, activation, expansion (proliferation), and/or differentiation of target cells in the composition. In some embodiments, the reagent contains a stimulatory agent that provides a primary activation signal to cells, wherein the stimulating agent contains at least one C binding partner (e.g., C1, C2, or C3, etc.), wherein the C binding partner binding is capable of reversibly binding to the Z1 binding site of the oligomeric reagent to reversibly bind the agent. In some embodiments, the reagent contains an additional agent that provides an additional signal to cells, wherein the additional agent contains at least one C binding partner (e.g., C1, C2, or C3, etc.), wherein the C binding partner capable of reversibly binding to the Z1 binding site of the oligomeric reagent to reversibly bind the agent. In some embodiments, the reagent contains a selective agent that specifically highlights binding to a particular cell surface molecule or marker, wherein the selective agent contains at least one C binding partner (e.g., C1, C2, or C3, etc.), when This binding partner C is capable of reversibly binding to the Z1 binding site of the oligomeric reagent to reversibly bind the agent.

In some embodiments, activation of the cells in the input composition is initiated during the process of contacting the cells of the input composition with an oligomeric protein reagent and/or viral particles. In such cases, the oligomeric protein reagent may have a receptor-binding agent immobilized thereon, for example, a stimulatory agent and/or an additional agent capable of inducing or modulating a signal in cells such as T cells. In some embodiments, the stimulatory agent comprises an MHC I:peptide complex or a functional portion thereof, an MHC II:peptide complex or a functional portion thereof, and/or is capable of delivering a stimulatory signal through a TCR/CD3 complex in a T cell, a CD3-containing complex in a T cell and/or an ITAM-containing molecule in a T cell. In some embodiments, the oligomeric reagent may have an additional agent immobilized thereon that is capable of providing an additional signal to cells, such as T cells. In some embodiments, the receptor-binding agent, eg, stimulatory agent and/or additional agent, is any agent as described herein, such as an anti-CD3 and/or anti-CD28 antibody (eg, Fab). Alternatively, a ligand, for example a natural ligand, of a receptor that triggers cell proliferation can also be used as a stimulatory agent. For example, the extracellular domain of CD19 can be used to cause the activation of intracellular signaling cascades of cells transduced to express a chimeric CD19 binding antigen receptor (CAR). In some embodiments, the oligomeric protein reagent (eg, streptavidin) is capable of both modulating and activating cell transduction, eg, stimulating cells during contact and optionally further incubation. In some embodiments, the binding of the oligomeric reagent containing the stimulating agent is reversible, eg in the presence of a competing agent, eg biotin.

In some embodiments, the present method can be used to selectively induce transduction and/or ex vivo expansion of specific cell populations, such as B cells, T cells, or natural killer cells. In some embodiments, the oligomeric protein reagent (e.g., streptavidin mutein) is a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, which may contain at least one selective agent reversibly associated with the same reagent, used to modulate transduction. In some embodiments, the oligomeric reagent (e.g., a streptavidin mutein) is a multimerization reagent and/or a particulate oligomeric reagent associated with one or more agents, which may contain a selective agent and one or both of the first or second receptor binding agents (e.g. , stimulating agent or additional agent) on the same reagent. In some embodiments, an oligomeric protein reagent (e.g., streptavidin), such as a multimerization reagent and/or a particulate oligomeric reagent coupled to one or more agents, is capable of both modulating cell transduction and preferably targeting transduction to a specific subset of selected or isolated cells. In some embodiments, an oligomeric protein reagent (e.g., streptavidin), such as a multimerization reagent and/or a particulate oligomeric reagent coupled to one or more agents, is capable of modulating cell transduction, e.g., preferably targeting transduction to a specific subset of selected or isolated cells , and activate, eg, stimulate cells, during contact and optionally further incubation.

In some embodiments, the binding of an oligomeric reagent containing binding agents, eg, a selective agent and/or a stimulating agent, is reversible, eg, in the presence of a competing agent, eg, biotin. As described below, in some aspects, the method includes adding or incubating a composition containing cells, viral particles, and an oligomeric reagent (e.g., a multimerization reagent and/or a particulate oligomeric reagent associated with one or more binding agents) with a competing agent to reverse alteration, dissociation, or decoupling of one or more binding agents from a cell or viral particles. In some embodiments, following redirection, dissociation, or disruption, one or more components of the composition can be removed, such as dissociating the oligomeric reagent, one or more coupling agents, and/or competing agent.

In some embodiments, the cells resulting from the present method (hereinafter also referred to as "output composition" or "incubated composition") include cells transduced with a viral vector, such as a viral vector containing nucleotides encoding a heterologous protein, such as a recombinant receptor, such as CAR. Heterologous in this context refers to a protein that is not normally expressed from a virus and/or is not encoded by the viral genome. In some embodiments, integration of a viral vector into the host genome can be assessed by measuring the level of expression of a recombinant protein, such as a heterologous protein encoded by a nucleic acid contained in the genome of the viral vector particles after incubation. A number of well known methods for assessing the level of expression of recombinant molecules can be used, such as detection by affinity based methods, eg immunoaffinity based methods, eg in the context of cell surface proteins, eg by flow cytometry. In some examples, expression is measured by detecting a transduction marker and/or a reporter construct. In some embodiments, a nucleic acid encoding a truncated surface protein is contained within a vector and is used as an expression and/or enhancement marker.

V. Compositions, Formulations and Methods of Administration

Also provided are compositions containing an engineered receptor (eg, an engineered antigen receptor), such as CAR or TCR, and compositions containing engineered cells, including pharmaceutical compositions and formulations. Also provided are methods of use and applications of the compositions, for example, for the treatment of diseases, conditions and disorders in which an antigen is expressed, or in methods of detection, diagnosis and prognosis.

A. Compositions/Preforms

The term "pharmaceutical formulation" refers to a preparation that is in such a form as to provide effective biological activity of the active ingredient contained therein, and which does not contain additional components that are unacceptably toxic to the subject to whom the resulting formulation will be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, adjuvant, stabilizer, or preservative.

In some aspects, the choice of carrier is determined in part by the specific cell and/or route of administration. Accordingly, there are many suitable ready-made forms. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol alcohol, butyl or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

in some aspects, the compositions contain buffering agents. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. the buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Known methods of obtaining input pharmaceutical compositions. illustrative methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease or condition being treated by the cells, preferably ingredients with activity complementary to the cell, where the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc. In some embodiments, the cells or antibodies are administered in the form of a salt, such as a pharmaceutically acceptable salt. Suitable pharmaceutically acceptable acid addition salts include salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic . gluconic, succinic and arylsulfonic acids, for example p-toluenesulfonic acid.

The active ingredients may be encapsulated in microcapsules, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. In some embodiments, the pharmaceutical composition is formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or as a liposome. Liposomes can serve to target host cells (eg, T cells or NK cells) to a particular tissue. Many methods are available for preparing liposomes, such as those described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and US Pat.

In a pharmaceutical composition, in some aspects, sustained release, delayed release, and sustained release delivery systems may be used such that delivery of the composition occurs before and with sufficient time to cause sensitization of the site to be treated. Many types of defined release delivery systems are available and known. Such systems can avoid re-administration of the composition, thereby increasing convenience for the subject and the clinician.

The pharmaceutical composition in some embodiments contains cells in amounts effective to treat or prevent a disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy is, in some embodiments, monitored by periodic evaluation of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until the desired suppression of the symptoms of the disease occurs. However, other dosage regimens may be useful and may be determined. The desired dose can be delivered by a single bolus of the composition, multiple bolus of the composition, or continuous infusion of the composition.

Cells can be administered using standard administration techniques, formulations and/or devices. Formulations and devices such as syringes and vials are provided for storing and administering the compositions. The introduction of cells can be autologous or heterologous. For example, immunoreactive cells or progenitors can be obtained from one subject and administered to the same subject or another compatible subject. Peripheral blood-derived immunoreactive cells or their progeny (obtained, for example, in vivo, ex vivo, or in vitro) can be administered by localized injection, including catheter injection, systemic injection, localized injection, intravenous injection, or parenteral administration. When a therapeutic composition (eg, a pharmaceutical composition containing a genetically modified immunoreactive cell) is administered, it will typically be formulated as a single dose injectable (solution, suspension, emulsion).

Formulations include formulations for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, cell populations are administered parenterally. the term "parenteral" within the scope of the invention includes intravenous, intramuscular, subcutaneous, rectal, vaginal and intraperitoneal administration. In some embodiments, cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.

Compositions in some embodiments are presented as sterile liquid preparations, eg, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous formulations, and solid formulations. In addition, liquid formulations are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions may be formulated within an appropriate viscosity range to provide longer periods of contact with specific tissues. Liquid or viscous compositions may contain carriers, which may be solvents or a dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

Sterile injectable solutions can be obtained by incorporating the cells into a solvent such as a mixture with a suitable carrier, diluent or adjuvant such as sterile water, saline, glucose, dextrose and the like. The compositions may also be lyophilized. The compositions may contain adjuvants such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosifying agents, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the formulation desired. In some aspects, standard guidelines can be consulted to obtain suitable formulations.

various additives may be added that enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. Prolonged absorption of the injectable pharmaceutical form can be achieved by the use of absorption delaying agents such as aluminum monostearate and gelatin.

sustained release formulations are available. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of molded articles such as films or microcapsules.

Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filter membranes.

B. Methods of administration

Methods for administering cells, populations, and compositions and uses of such cells, populations, and compositions for the treatment or prevention of diseases, conditions, and disorders, including cancer, are provided. In some embodiments, cells, populations, and compositions are administered to a subject or patient having a particular disease or condition being treated, for example, through adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, the cells and compositions produced by the present methods, such as engineered compositions and final production compositions after incubation and/or other processing steps, are administered to a subject, such as a subject having or at risk of a disease or condition. In some aspects, the methods thus treat, for example, alleviate one or more symptoms of a disease or condition, for example, by reducing tumor mass in a cancer that expresses an antigen recognized by the engineered T cell.

Methods for introducing cells for adoptive cell therapy are known and can be used in combination with the present methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in US Patent Application Publication No. 2003/0170238 Gruenberg et al; US Pat. No. 4,690,915 Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

Within the meaning of the invention, a "subject" is a mammal, such as a human or other animal, and usually a human. In some embodiments, the subject, eg, the patient, to whom the cells, cell populations, or compositions are administered is a mammal, usually a primate, such as a human. In some embodiments, the primate is an ape or great ape. The subject may be male or female, and may be of any suitable age, including infants, adolescents, young adults, adults, and the elderly. In some embodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, "treatment" (and its grammatical variants such as "treat") refers to the total or partial amelioration or reduction of a disease or condition or disorder, or a symptom, an adverse effect or outcome, or an associated phenotype. Desirable treatment outcomes include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or mitigation of the disease state, and remission or improvement in prognosis. The terms do not imply complete cure of a disease or complete elimination of any symptoms or effect(s) on all symptoms or results.

In the context of the invention, "delaying the development of a disease" means delaying, delaying, slowing down, inhibiting, stabilizing, suppressing and/or postponing the development of a disease (such as cancer). This delay may be of varying length of time depending on the history of the disease and/or the individual being treated. As is apparent to one of skill in the art, a sufficient or significant delay may, in fact, involve prophylaxis, as long as the individual does not develop the disease. For example, it is possible to delay the late stage of cancer, such as the development of metastasis.

"Prevention" within the meaning of the invention includes providing prophylaxis against the occurrence or recurrence of a disease in a subject who may be predisposed to the disease but in whom the disease has not yet been diagnosed. In some embodiments, the provided cells and compositions are used to delay the progression of a disease or to slow the progression of a disease.

In the context of the invention, to "suppress" a function or activity means to reduce the function or activity compared to other such states except for the state or parameter of interest, or alternatively compared to another state. For example, cells that inhibit tumor growth reduce the rate of tumor growth compared to the rate of tumor growth in the absence of cells.

An "effective amount" of an agent, such as a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an amount effective in dosages/amounts and for periods of time necessary to achieve the desired result, such as a therapeutic or prophylactic result.

A "therapeutically effective amount" of an agent, such as a pharmaceutical formulation or cells, refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result, for example, to treat a disease, condition or disorder and/or pharmacokinetic or pharmacodynamic treatment result. A therapeutically effective amount may vary depending on such factors as disease state, age, sex and weight of the subject and the population of cells administered. In some embodiments, the methods provided include administering the cells and/or compositions in effective amounts, such as therapeutically effective amounts.

"Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Usually, but not necessarily, since a prophylactic dose is used in subjects before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The disease or condition being treated can be any in which antigen expression is associated with and/or involved in the etiology of the disease state or disorder, eg, causes, exacerbates or is otherwise involved in such disease, condition or disorder. Exemplary diseases and conditions may include diseases or conditions associated with cancer or cell transformation (eg, cancer), an autoimmune or inflammatory disease, or an infectious disease, eg, caused by a bacterial, viral, or other pathogen. Illustrative antigens, which include antigens associated with various diseases and conditions that can be treated, are described above. In specific embodiments, the chimeric antigen receptor or transgenic TCR specifically binds to an antigen associated with a disease or condition.

Thus, the provided methods and uses include methods and uses for adoptive cell therapy. In some embodiments, the methods include administering cells or a composition containing cells to a subject, tissue or cell, for example, at risk or suspected of having a disease, condition or disorder. In some embodiments, cells, populations, and compositions are administered to a subject having a particular disease or condition being treated, for example, through adoptive cell therapy, such as adoptive T cell therapy. In some embodiments, cells or compositions are administered to a subject, such as a subject having or at risk of a disease or condition, alleviate one or more symptoms of the disease or condition.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by autologous transfer in which cells are isolated and/or otherwise obtained from a subject to receive cell therapy or from a sample obtained from such a subject. Thus, in some aspects, cells are obtained from a subject, such as a patient in need of treatment, and the cells, after isolation and processing, are administered to the same subject.

In some embodiments, cell therapy, such as adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and/or otherwise obtained from a subject other than the subject to receive or who ultimately receives cell therapy, for example , the first subject. In such embodiments, the cells are then administered to another subject, such as a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses an HLA of the same class or supertype as the first subject. Cells can be administered by any suitable method. The route of administration and doses may depend in part on whether the administration is transient or continuous. Various treatment regimens include, but are not limited to, single or multiple administration at different time points, bolus administration, and pulsed infusion.

In some embodiments, cells or distinct populations of cell subtypes are administered to a subject in the range of from about one million to about 100 billion cells and/or as many cells per kilogram of body weight as, for example, from 1 million to about 50 billion cells (e.g., about 5 million cells, approximately 25 million cells, approximately 500 million cells, approximately 1 billion cells, approximately 5 billion cells, approximately 20 billion cells, approximately 30 billion cells, approximately 40 billion cells, or a range limited by any two of the above values), e.g. , from about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range limited by any two of the above values), and in some cases from approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 250 million cells, approximately 350 million cells, approximately 450 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 3 billion cells, approximately 30 billion cells , approximately 45 billion cells) or any value between these ranges and/or per kilogram of body weight. Again, dosages may vary depending on the characteristics specific to the disease or disorder and/or the patient and/or other treatments. In some embodiments, cells are administered as part of a combination treatment, eg, simultaneously or sequentially, in any order, with another therapeutic intervention, such as an antibody or engineered cell, or a receptor or agent, such as a cytotoxic or therapeutic agent. The cells, in some embodiments, are administered in conjunction with one or more additional therapeutic agents, or in combination with another therapeutic intervention, either simultaneously or sequentially in any order. In some instances, the cells are co-administered with another therapy close enough in time that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, cells are administered prior to the administration of one or more additional therapeutic agents. In some embodiments, the cells are administered following the administration of one or more additional therapeutic agents. In some embodiments, the implementation of one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods include administering a chemotherapeutic agent.

Following cell administration, the biological activity of the engineered cell populations is measured in some embodiments, for example, by any of a number of known methods. Evaluation parameters include specific binding of the engineered or native T cell or other immune cell to the antigen in vivo, eg by imaging, or ex vivo, eg by ELISA or flow cytometry. In some embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32 (7): 689- 702 (2009) and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004). In some embodiments, the biological activity of cells is measured by analyzing the expression and/or secretion of one or more cytokines such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by evaluating a clinical outcome, such as a reduction in tumor mass or burden.

In certain embodiments, engineered cells are further modified in any number of ways such that their therapeutic or prophylactic efficacy is increased. For example, an engineered CAR or TCR expressed by a population can be conjugated either directly or indirectly via a linker to a targeting moiety. The practice of conjugating compounds, such as CAR or TCR, with targeting moieties is known in the art. See, for example, Wadwa et al., J. Drug Targeting 3:11 (1995) and U.S. Patent 5,087,616.

VI. Definitions

Within the scope of the invention, the singular forms include references to the plural, unless the context clearly dictates otherwise. For example, the singular forms mean "at least one" or "one or more". It is understood that the aspects and variants described herein include "consisting of" and/or "consisting essentially of" the aspects and variants.

Throughout this disclosure, various aspects of the claimed subject matter are presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values within that range. For example, when a range of values is presented, every intermediate value between the upper and lower limits of that range and any other specified or intermediate value within the specified range is intended to be within the claimed subject matter. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also covered by the claimed subject matter, subject to any specifically excluded limit within the specified range. If the specified range includes one or both of these limits, ranges excluding one or both of these included limits are also included in the claimed subject matter. This applies regardless of the latitude of the range.

The term "approximately" in the context of the present invention refers to the usual range of errors for the corresponding value, known to a person skilled in the art. Reference to an "approximate" value or parameter in this document includes (and describes) embodiments that are directed to that value or parameter as such. For example, a description referring to "about X" includes a description of "X". In specific embodiments, "about" is ±25%, ±20%, ±15%, ±10%, ±5%, ±1%, ±0.5%, ±0.1%, ±0.01%, or ±0.001%.

In the context of the invention, a composition refers to any mixture of two or more products, substances or compounds, including cells. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, "enrichment" when referring to one or more specific cell types or cell populations refers to an increase in the number or percentage of a cell type or population, for example, compared to the total number of cells in a composition or volume of a composition, or relative to other cell types, for example , by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present in the population of cells or on the cell to be depleted. the term does not require complete removal of other cells, cell types or populations from the composition, and does not require enriched cells to be present in or even close to 100% enriched composition.

Within the scope of the invention, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in the cell of a particular marker, usually a surface marker. When referring to a surface marker, the term refers to the presence of surface expression detectable by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, where the staining is detected by flow cytometry at a level substantially greater than the detected staining, performing the same procedure with an isotype-like control under otherwise identical conditions and/or at a level substantially similar to that of a cell known to be positive for the marker and/or at a level substantially higher than that of a cell known to be as negative for the marker.

Within the scope of the invention, a statement that a cell or population of cells is "negative" for a particular marker refers to the absence of a significant detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression detectable by flow cytometry, e.g. by staining with an antibody that specifically binds to the marker and detecting said antibody, where the staining is not detected by flow cytometry at a level substantially greater than the detected staining. by performing the same procedure with an isotype-like control under otherwise identical conditions and/or at a level substantially lower than that of a cell known to be positive for the marker and/or at a level substantially similar to the cell , which is known to be negative for the marker.

The term "expression", as used herein, refers to the process by which a polypeptide is derived from the coding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

Within the scope of the invention, a subject includes any living organism, such as humans and other mammals. Mammals include, but are not limited to, humans, and non-human animals, including farm animals, sport animals, rodents, and pets.

For the purposes of the invention, a control refers to a sample that is essentially identical to the test sample, except that it is not treated with the test parameter, or if it is a plasma sample, it may be from a normal volunteer without the condition of interest. control can also be internal control.

VII. Exemplary Embodiments

Among the provided embodiments:

1. A particulate oligomeric reagent containing a plurality of streptavidin or streptavidin mutein molecules, wherein the particle size of the oligomeric reagent is i) a radius greater than 25 nm, ii) a molecular weight of at least 5×10 ⁶ g/mol; and/or (iii) at least 100 tetramers of streptavidin or streptavidin mutein per oligomeric particulate reagent.

2. The particulate oligomeric reagent of Embodiment 1, wherein the streptavidin or streptavidin mutein molecules bind or are capable of binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and/or a biologically active fragment or streptavidin binding peptide thereof.

3. The particulate oligomeric reagent of embodiment 2, wherein the streptavidin or streptavidin mutein molecules reversibly bind or are capable of reversibly binding to biotin, avidin, a biotin analog or mutein, an avidin analog or mutein, and/or a biologically active fragment or streptavidin-binding fragment thereof. peptide.

4. The particulate oligomeric reagent according to any one of embodiments 1-3, wherein the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules, wherein the streptavidin mutein molecules comprise the amino acid sequence Va1 ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or lle ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44-47 with reference to positions in streptavidin in the amino acid sequence shown in SEQ ID NO: 1.

5. The particulate oligomeric reagent according to any one of embodiments 1-4, wherein the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that comprise:

a) the amino acid sequence given in any of SEQ ID NOs: 3-6, 27, 28, 60 or 61;

b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to any SEQ ID NO: 3-6, 27, 28, 60 or 61 and contain the amino acid sequence corresponding to Va1⁴⁴-Thr⁴⁵-ala⁴⁶-Arg⁴⁷or lle⁴⁴-Gly⁴⁵-ala⁴⁶-Arg⁴⁷, and/or bind reversibly to biotin or its biologically active form, analog or mutein of biotin or their biologically active fragment or streptavidin-binding peptide; or

c) a functional fragment of a) or b) that reversibly binds to biotin or its biologically active form, biotin analogue or mutein or their biologically active fragment or streptavidin-binding peptide.

6. The particulate oligomeric reagent according to any one of embodiments 1-5, wherein the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that contain the amino acid sequence shown in SEQ ID NO: 6 or 61.

7. The particulate oligomeric reagent according to any one of embodiments 4-6, wherein the streptavidin mutein molecule further comprises an amino acid substitution or substitutions at position corresponding to 117, 120 and/or 121 with reference to positions in streptavidin in the amino acid sequence given in SEQID NO:1.

8. The particulate oligomeric reagent of Embodiment 7, wherein:

the amino acid substitution or substitutions are selected from Glu117, Asp117, Arg117, Ser120, Ala120, Gly120, Trp121, Tyr121 or Phe121; or

the amino acid substitution or substitutions are selected from one or more of Glu117, Gly120 or Tyr121; or

amino acid substitutions are selected from Glu117, Gly120 or Tyr121.

9. The particulate oligomeric reagent according to any one of embodiments 1-8, wherein the particulate oligomeric reagent comprises a plurality of streptavidin mutein molecules that comprise:

a) the amino acid sequence given in SEQ ID NO: 27 or 28;

b) an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to SEQ ID NO: 28 and contains the amino acid sequence corresponding to Va1 ⁴⁴ , Thr ⁴⁵ , Ala ⁴⁶ , Arg ⁴⁷ , Glu ¹¹⁷ , Gly ¹²⁰ and Tyr ¹²¹ , and/or reversibly binds to biotin or a biologically active fragment, analog or mutein of biotin or their biologically active fragment or streptavidin-binding peptide; or

c) a functional fragment of a) or b) that reversibly binds to biotin or a biologically active fragment, analog or mutein of biotin or their biologically active fragment or streptavidin-binding peptide.

10. A particulate oligomeric reagent according to any one of embodiments 1-9, wherein the particulate oligomeric reagent is bound or capable of binding to one or more agents.

11. The particulate oligomeric reagent of embodiment 10, wherein the one or more agents comprise a binding partner, wherein the binding partner is capable of binding, optionally reversibly binding, to one or more binding sites on the oligomeric particulate reagent.

12. The particulate oligomeric reagent of Embodiment 11, wherein the binding partner comprises a streptavidin binding peptide.

13. The particulate oligomeric reagent of embodiment 11 or 12, wherein the binding partner comprises a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO : 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp -Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro -Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

14. A particulate oligomeric reagent according to any one of embodiments 10-13, wherein one or more agents bind or are able to bind to a molecule expressed on the surface of a target cell.

15. A particulate oligomeric reagent according to any one of embodiments 10-14, wherein one or more of the agents is or contains an antibody or an antigen-binding fragment thereof.

16. The particulate oligomeric reagent of embodiment 15, wherein one or more of the reagents is or contains a monovalent antibody fragment.

17. The particulate oligomeric reagent of Embodiment 15 or Embodiment 16, wherein one or more of the agents is or contains a Fab.

18. A particulate oligomeric reagent according to any one of embodiments 10-17, wherein the one or more agents is a receptor binding agent that binds or is capable of binding to a receptor expressed on the surface of a target cell.

19. A particulate oligomeric reagent according to any one of embodiments 10-18, wherein the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, wherein the binding induces or modulates a signal in the target cell .

20. The particulate oligomeric reagent according to embodiment 18 or item 19, wherein the target cell is an immune cell.

21. A particulate oligomeric reagent according to any one of embodiments 18-20, wherein the target cell is a T cell.

22. The particulate oligomeric reagent of any one of embodiments 18-21, wherein the receptor binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3.

23. The particulate oligomeric reagent of embodiment 22, wherein the stimulatory agent is a first receptor binding agent and the particulate oligomeric reagent comprises a second receptor binding agent, wherein the second receptor binding agent is capable of specifically binding to a second molecule on the surface of the target cell, this binding to the second molecule being optionally capable of inducing or modulating a signal in the target cells.

24. The particulate oligomeric reagent of Embodiment 23, wherein the second receptor binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is is or contains an adhesion molecule or a factor that induces cytokine production, chemokine production and/or expression of an adhesion molecule.

25. The particulate oligomeric reagent of Embodiment 22 or Embodiment 23, wherein the second receptor binding agent specifically binds to a costimulatory molecule and the costimulatory molecule is CD28.

26. A particulate oligomeric reagent according to any one of embodiments 10-25, wherein one or more of the agents is an anti-CD3 antibody and an anti-CD28 antibody, optionally an anti-CD3 Fab and an anti-CD28 Fab.

27. The particulate oligomeric reagent according to any one of embodiments 18-21, wherein the receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family, or is a TNF family receptor, cytokine receptor, chemokine the receptor is or is or contains an adhesion molecule or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

28. The particulate oligomeric reagent according to any one of embodiments 18-21, 23, or 24, wherein the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40 or HVEM.

29. The particulate oligomeric reagent according to any one of embodiments 18-21, 23, or 24, wherein the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor and the cytokine receptor is selected from IL-2R, IL- 1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2.

30. A particulate oligomeric reagent according to any one of embodiments 18-21, 23, or 24, wherein the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4.

31. The particulate oligomeric reagent according to any one of embodiments 18-21, 23, or 24, wherein the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand, which specifically binds to a cytokine or chemokine receptor.

32. The particulate oligomeric reagent of Embodiment 31, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein

ligand specifically binds IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR type I, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2 ; and/or

the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or it is one of them biologically active fragment.

33. The particulate oligomeric reagent of Embodiment 30, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein

the ligand binds specifically to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or

the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof.

34. The particulate oligomeric reagent according to any one of embodiments 18-21, 23, or 24, wherein the receptor binding agent (second receptor binding agent) is an adhesion molecule and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is a biologically active fragment thereof.

35. A particulate oligomeric reagent according to any one of embodiments 10-34, wherein one or more agents is a selective agent, wherein the selective agent binds or is able to bind to a selectable marker that is expressed on the surface of the target cell.

36. The particulate oligomeric reagent of Embodiment 35, wherein the target cell is an immune cell.

37. The particulate oligomeric reagent of Embodiment 35 or Embodiment 36, wherein the target cell is a lymphocyte or an antigen presenting cell.

38. A particulate oligomeric reagent according to any one of embodiments 35-37, wherein the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell.

39. A particulate oligomeric reagent according to any one of embodiments 35-38, wherein the target cell is a T cell.

40. A particulate oligomeric reagent according to any one of embodiments 35-39, wherein the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

41. The particulate oligomer reagent according to any one of embodiments 1-40, wherein the particulate oligomer reagent has a radius greater than 25 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, or more than 90 nm.

42. The particulate oligomer reagent according to any one of embodiments 1-41, wherein the particulate oligomer reagent has a radius between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm or between 90 nm and 110 nm inclusive, or 90 nm ±15 nm or 95 nm ± 20-25 nm.

43. The particulate oligomeric reagent according to any one of embodiments 1-42, wherein the particulate oligomeric reagent has a radius of less than 150 nm.

44. The particulate oligomeric reagent according to any one of embodiments 41-43, wherein the radius is the hydrodynamic radius.

45. The particulate oligomeric reagent according to any one of embodiments 1-44, wherein the particulate oligomeric reagent has a molecular weight of at least 1×10 ⁷ g/mol, at least 5×10 ⁷ g/mol, or at least measure 1×10 ⁸ g/mol.

46. The particulate oligomeric reagent according to any of embodiments 1-45, wherein the particulate oligomeric reagent has a molecular weight between 1×10 ⁶ g/mol and 1×10 ¹⁰ g/mol, between 1×10 ⁷ g/mol mol and 1×10 ⁹ g/mol, between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g /mol and 2×10 ⁸ g/mol.

47. A particulate oligomeric reagent according to any one of embodiments 1-46, wherein the particulate oligomeric reagent comprises at least 100 streptavidin or streptavidin mutein tetramers, at least 500 streptavidin or streptavidin mutein tetramers, at least 1000 streptavidin tetramers or a streptavidin mutein, at least 1500 tetramers of streptavidin or streptavidin mutein, or at least 2000 tetramers of streptavidin or streptavidin mutein.

48. A particulate oligomeric reagent according to any one of embodiments 1-47, wherein the particulate oligomeric reagent comprises between 100 and 50,000 streptavidin or streptavidin mutein tetramers, between 1,000 and 20,000 streptavidin or streptavidin mutein tetramers, between 1,000 and 10,000 streptavidin tetramers or streptavidin mutein or between 2000 and 5000 tetramers of streptavidin or streptavidin mutein.

49. The particulate oligomeric reagent according to any one of embodiments 1-48, wherein a plurality of streptavidin or streptavidin mutein molecules contain lysine residues, wherein less than 20%, 10%, 5%, 1% of the lysine residues contain an N-substituted iminothiolane .

50. A composition comprising one or more particulate oligomeric reactants according to any one of Embodiments 1-49.

51. The composition of Embodiment 50, wherein the one or more particulate oligomeric reactants is a plurality of particulate oligomeric reactants.

52. The composition of Embodiment 51, wherein the plurality of particulate oligomeric reactants have i) an average radius greater than 70 nm; ii) an average molecular weight of at least 1×10 ⁸ g/mol; and/or iii) an average amount of streptavidin or streptavidin tetramers per particulate oligomeric reagent of at least 2000 and/or iv) a radius size distribution in which at least 95% of the plurality of particulate oligomeric reagents have a radius between 10 nm and 150 nm.

53. The composition of Embodiment 51 or 52, wherein the plurality of particulate oligomeric reactants have an average radius greater than 25 nm, greater than 50 nm, greater than 60 nm, greater than 70 nm, greater than 80 nm, greater than 90 nm or more than 100 nm.

54. A composition according to any one of embodiments 51-53, wherein:

a plurality of particulate oligomeric reagents have an average radius between 25 nm and 150 nm, between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 110 nm, or between 90 nm and 110 nm, inclusive; or

a plurality of particulate oligomeric reagents have an average radius of 90 nm±15 nm, 95 nm±20-25 nm, or 97±10 nm.

55. A composition according to any one of embodiments 51-54, wherein at least 95% of the plurality of particulate oligomeric reactants have a radius between 50 and 150 nm, between 70 nm and 140 nm, between 80 nm and 120 nm, between 80 nm and 115 nm, between 80 nm and 100 nm, between 90 nm and 110 nm and/or between 100 nm and 120 nm.

56. A composition according to any one of embodiments 51-55, wherein at least 95% of the particulate oligomeric reactants have a radius within ±50%, ±25%, ±20%, ±15%, ±10% and/or ± 5% of the mean and/or median radius of a plurality of particulate oligomeric reagents.

57. A composition according to any one of embodiments 51-56, wherein the plurality of particulate oligomeric reactants have an average radius between 80 nm and 115 nm, and wherein at least 95% of the particulate oligomeric reactants have a radius within ±25 % average radius.

58. A composition according to any one of embodiments 51-57, wherein the plurality of particles have an average molecular weight between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g/mol and 2×10 ⁸ g/mol, inclusive.

59. A composition according to any one of embodiments 51-58, wherein the plurality of particulate oligomeric reagents comprises an average amount of streptavidin or streptavidin tetramers per particulate oligomeric reagent of at least 100, at least 500, at least 1000, at least at least 1500 or at least 2000.

60. A composition according to any one of embodiments 51-59, wherein the plurality of particulate oligomer reagents comprises an average amount of streptavidin or streptavidin tetramers per particulate oligomer reagent between 100 and 50,000, between 1,000 and 20,000, between 1,000 and 10,000, or between 2,000 and 5000, always inclusive.

61. A composition according to any one of embodiments 51-60, wherein the average radius of the plurality of oligomeric particles does not increase by more than 25% or 10% when stored at or below -80°C, at or below -20°C, and/ or at or below 4° C. for at least 1, 3, 9, 27, or 46 weeks.

62. A composition according to any one of embodiments 51-61, wherein the average radius of the plurality of oligomeric particles does not increase by more than 10% when stored at or below about 4°C for at least one week.

63. A composition according to any one of embodiments 61-62, wherein the average radius of the plurality of oligomeric particles does not increase by more than 10% when stored at or below about 4°C for at least 3 weeks.

64. A composition according to any one of items 51-63, wherein the average radius of the plurality of oligomeric particles does not increase by more than 10% when stored at or below about 4° C. for at least 9 weeks.

65. A method for preparing a particulate oligomeric reagent containing streptavidin or a streptavidin mutein, the method comprising:

incubating a plurality of activated streptavidin or streptavidin mutein molecules containing a thiol-reactive functional group capable of reacting with a thiol functional group and a plurality of thiolated streptavidin or streptavidin mutein molecules containing one or more thiol functional group, thereby obtaining a particle composition containing oligomeric particles streptavidin or streptavidin mutein;

separating oligomeric particles from monomeric and/or smaller oligomeric molecules; and

contacting the oligomeric particles with a stabilizing agent, thereby obtaining a particulate oligomeric reagent.

66. The method of Embodiment 65, wherein the plurality of activated streptavidin or streptavidin mutein molecules are generated by incubating the first plurality of streptavidin or streptavidin mutein molecules with an activating agent that is capable of converting one or more amines to a thiol-reactive functional group.

67. The method according to embodiment 65 or embodiment 66, wherein the plurality of thiolated streptavidin or streptavidin mutein molecules is created by incubating the second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent that adds or is capable of adding a thiol functional group to one or more lysine residues.

68. A method for producing oligomeric reagents in the form of particles, and the method includes:

(a) incubating a first plurality of streptavidin or streptavidin mutein molecules with an activating agent under conditions to convert one or more amines into a thiol-reactive group capable of reacting with a thiol functional group, thereby generating a plurality of activated streptavidin or streptavidin mutein molecules;

(b) incubating a second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent that adds or is capable of adding a thiol functional group to one or more lysine residues, thereby creating a plurality of thiolated streptavidin or streptavidin mutein molecules; and

(c) incubating a plurality of activated streptavidin or streptavidin mutein molecules with a plurality of thiolated streptavidin or streptavidin mutein molecules, thereby obtaining a particulate composition comprising particulate oligomeric reagents;

wherein the method is carried out under conditions in which, at the start of the incubation in (c), the plurality of thiolated streptavidin or streptavidin mutein molecules are such that, on average, at least 60% of the lysines contain a thiol functional group, and/or, on average, at least 10 lysines on a thiolated streptavidin tetramer or streptavidin mutein contain a thiol functional group.

69. The method of Embodiment 68, further comprising separating the particulate oligomeric reagents from monomeric and/or smaller oligomeric streptavidin or streptavidin mutein molecules.

70. The method according to any one of embodiments 65-69, wherein the incubation of the first plurality of streptavidin or streptavidin mutein molecules with the activating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to activating reagent between 1:1 and 1:10.

71. The method according to any of embodiments 66-70, wherein the incubation of the first plurality of streptavidin or streptavidin mutein molecules with the activating agent is carried out at a molar ratio of streptavidin or streptavidin mutein to activating reagent of 1:2±2%.

72. The method according to any one of embodiments 66-71, wherein the activating agent comprises a heterobifunctional crosslinker.

73. The method according to any one of embodiments 66-72, wherein the activating agent comprises sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo SMCC) and/or succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH ).

74. The method according to any one of embodiments 65-73, wherein the thiol-reactive functional group is a haloacetyl group, a maleimide group, an aziridine group, an acryloyl group, an arylizing agent, a vinylsulfone group, a pyridyl disulfide, a TNB-thiol, or a disulfide reducing agent.

75. The method according to any one of embodiments 65-74, wherein the thiol-reactive functional group is a maleimide group.

76. The method according to any one of embodiments 65-75, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at neutral pH.

77. The method according to any one of embodiments 65-76, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a pH between 6.8 and 7.5.

78. The method according to any one of embodiments 65-77, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a pH between 7.0 and 7.4, optionally at or about 7.2.

79. The method according to any one of embodiments 65-78, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at a temperature between 4°C and 39°C.

80. The method according to any one of embodiments 65-79, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated at room temperature, optionally between 20°C and 25°C, optionally at about 23°C or about 24°C .

81. The method according to any one of embodiments 65-80, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated for 15 minutes to 6 hours or 30 minutes to 2 hours, always inclusive.

82. The method according to any one of embodiments 65-81, wherein the first plurality of streptavidin or streptavidin mutein molecules and the activating agent are incubated for 45 minutes to 1.5 hours, inclusive, optionally within or about 1 hour.

83. The method according to any one of embodiments 66-82, wherein the incubation of the second plurality of streptavidin or streptavidin mutein molecules with the thiolating agent is carried out at a molar ratio of thiolating reagent to each primary amine between 10:1 and 1:1, inclusive, per streptavidin molecule or streptavidin mutein.

84. The method according to any one of embodiments 66-83, wherein the incubation of the second plurality of streptavidin or streptavidin mutein molecules with the thiolating agent is carried out at a molar ratio between 1:50 and 1:500, inclusive, of the streptavidin tetramer or streptavidin mutein to the thiolating agent.

85. The method according to any one of embodiments 66-84, wherein the incubation of the second plurality of streptavidin or streptavidin mutein molecules with the thiolating agent is carried out at a molar ratio of or about 1:100 of the streptavidin tetramer or streptavidin mutein to the activating agent.

86. The method according to any one of embodiments 66-85, wherein the thiolating agent is or contains 2-iminothiolane.

87. The method according to any one of embodiments 66-86, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated at pH 7.0 or greater, optionally between 7.0 and 8.0, inclusive.

88. The method according to any one of embodiments 66-87, wherein the second plurality of streptavidin or streptavidin mutein molecules and a thiolating agent are incubated at a pH of approximately 7.7.

89. The method according to any one of embodiments 66-88, wherein the incubation of the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent is started in the presence of a buffer with a pH of 8.0 or greater, optionally between 8.0 and 9.0, inclusive.

90. The method according to any one of embodiments 66-89, wherein the incubation of the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent is started in the presence of a buffer with a pH of or about 8.5.

91. The method according to embodiment 89 or 90, wherein the buffer contains a borate.

92. The method according to any of embodiments 89-91, wherein the buffer contains 10 mM-200 mM borate or 50 mM-100 mM borate, always inclusive, optionally about 100 mM borate.

93. The method according to any one of embodiments 65-92, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated at a temperature between 4°C and 39°C.

94. The method according to any one of embodiments 65-93, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated at room temperature, optionally between 20°C and 25°C, optionally at or about 23°C, or at or at approximately 24°C.

95. The method according to any one of embodiments 66-94, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated for 15 minutes to 2 hours, or 15 minutes to 1.5 hours.

96. The method according to any one of embodiments 66-95, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated for 15 minutes to 2 hours or 25 minutes to 1 hour, always inclusive.

97. The method according to any one of embodiments 66-96, wherein the second plurality of streptavidin molecules and the thiolating agent are incubated for or about 1 hour.

98. The method according to any one of embodiments 66-97, wherein the second plurality of streptavidin or streptavidin mutein molecules and the thiolating agent are incubated for or about 25 minutes.

99. The method according to any one of embodiments 65-98, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are in a 1:X molar ratio during incubation, wherein X is the amount of lysine residues available for thiolation, per streptavidin molecule or streptavidin mutein.

100. The method according to any one of embodiments 65-99, wherein the molar ratio is 1:1 to 1:8, or 1:2 to 1:6, optionally equal to or approximately equal to 1:4.

101. The method according to any one of embodiments 65-100, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a pH between 6.8 and 7.5, inclusive.

102. The method according to any one of embodiments 65-101, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a pH between 7.0 and 7.4, inclusive.

103. The method according to any one of embodiments 65-102, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at pH or about 7.2.

104. The method according to any one of embodiments 65-103, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at a temperature between 4°C and 39°C, inclusive.

105. The method according to any one of embodiments 65-104, wherein the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules are incubated at room temperature, optionally between 20°C and 25°C, inclusive, optionally at or about at 23°C or at or about 24°C.

106. The method according to any one of embodiments 65-105, wherein the plurality of activated streptavidin molecules and the plurality of thiolated streptavidin molecules are incubated for 15 minutes to 6 hours or 30 minutes to 2 hours, always inclusive.

107. The method according to any one of embodiments 65-106, wherein the plurality of activated streptavidin molecules and the plurality of thiolated streptavidin molecules are incubated for 45 minutes to 1.5 hours, inclusive, optionally within or about 1 hour.

108. The method according to any one of embodiments 65-107, wherein the incubation of the activated streptavidin or streptavidin mutein molecules and the thiolated streptavidin or streptavidin mutein molecules is terminated by contacting the molecules with N-ethylmaleimide (NEM).

109. The method according to any one of embodiments 68-108, wherein at least part of the incubation of the first plurality of streptavidin molecules or streptavidin mutein with an activating agent and at least part of the incubation of the second plurality of streptavidin molecules or streptavidin mutein with a thiolating agent are carried out separately in one and the same time.

110. The method according to embodiment 109, wherein the incubation of the first plurality of streptavidin or streptavidin mutein molecules with an activating agent and the incubation of the second plurality of streptavidin or streptavidin mutein molecules with a thiolating agent are carried out for substantially the same period of time and/or are performed essentially at the same time.

111. The method according to any one of embodiments 68-110, wherein prior to incubation of the thiolated streptavidin or streptavidin mutein molecules and the activated streptavidin or streptavidin mutein molecules, the method comprises:

(i) removing the activating agent from the composition containing the activated streptavidin or streptavidin mutein molecules; and/or

(ii) removing the thiolating agent from the composition containing the thiolated streptavidin or streptavidin mutein molecules.

112. The method according to any one of embodiments 68-111, wherein the incubation of the plurality of activated streptavidin or streptavidin mutein molecules and the plurality of thiolated streptavidin or streptavidin mutein molecules is started within 15 minutes after the end of incubation of the second plurality of streptavidin molecules with the thiolating agent and/or after removal a thiolating agent from a composition containing thiolated streptavidin or streptavidin mutein molecules.

113. A method for producing oligomeric reagents in the form of particles, including:

incubation of the first plurality of streptavidin or streptavidin mutein molecules with succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH) for or about 1 hour at pH equal to or approximately equal to 7.2, thereby creating a plurality of activated streptavidin or mutein molecules streptavidin containing a maleimide thiol-reactive functional group;

incubating a second plurality of streptavidin or streptavidin mutein molecules with 2-iminothiolane for or about 1 hour at a pH between 7.5 and 8.5, inclusive, thereby creating a plurality of thiolated streptavidin molecules containing one or more thiol functional groups; and

incubating a plurality of activated streptavidin molecules or a streptavidin mutein with a plurality of thiolated streptavidin molecules for or about 1 hour at a pH of or about 7.2, thereby obtaining a particulate composition comprising oligomeric particulate reagents;

wherein the incubation of the plurality of activated streptavidin molecules with the plurality of thiolated streptavidin molecules is started within 10 minutes after the incubation of the second plurality of streptavidin molecules with 2-iminothiolane ends.

114. The method according to any one of embodiments 68-113, wherein the method further comprises contacting particulate oligomeric reactants with a stabilizing agent.

115. The method according to any one of embodiments 65-67 or 114, wherein the stabilizing agent reduces the amount of N-substituted iminothiolane present on the lysine residues of the particulate oligomeric reagents.

116. The method according to any one of embodiments 65-67 or 114-115, wherein the stabilizing agent reduces the amount of N-substituted iminothiolane present on the lysine residues of the particulate oligomeric reagents by at least 25%, at least 50%, by at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

117. The method according to any of embodiments 65-67 and 114-116, wherein the stabilizing agent contains hydroxylamine.

118. The method according to any one of embodiments 65-67 and 114-117 wherein the stabilizing agent is removed from the particulate oligomeric reagents by chromatography, optionally by size exclusion chromatography (SEC).

119. The method of any one of embodiments 65-118, wherein the particulate oligomeric reactants have a radius of less than 150 nm.

120. The method of any one of embodiments 65-119, further comprising filter sterilization of the particulate oligomeric reagents.

121. The method according to any one of embodiments 65-67 and 69-120, wherein the particulate oligomeric reagents are separated from monomeric or smaller oligomeric streptavidin or streptavidin mutein molecules by size exclusion chromatography.

122. The method of Embodiment 113, wherein the exclusion limit is more than or about more than 100 kDa, 500 kDa, 750 kDa, 1000 kDa, or 2000 kDa.

123. The method according to embodiment 121 or embodiment 122, wherein the exclusion limit is from or about 500 kDa to 1000 kDa.

124. The method according to any one of embodiments 121-123, wherein the exclusion limit is or is about 75 kDa.

125. The method according to any one of embodiments 65-67 and 69-124, comprising collecting one or more fractions containing free volume, thereby separating particulate oligomeric reagents from monomeric or smaller oligomeric streptavidin or streptavidin mutein molecules.

126. The method of any one of embodiments 65-125, further comprising storing the particulate oligomeric reactants at or below 4°C, at or below -20°C, or at or below -80°C.

127. The method of any one of embodiments 65-126, further comprising mixing the particulate oligomeric reactants with one or more agents under conditions to reversibly bind the one or more particulate oligomeric reactants.

128. Particulate oligomeric reactant prepared by the method of any of embodiments 65-127.

129. A method for multimerizing one or more agents with a particulate oligomeric reagent, the method comprising mixing the particulate oligomeric reagent according to any one of paragraphs. 1-49, compositions containing oligomeric reagent in the form of particles according to any one of paragraphs. 50-64 or a particulate oligomeric reagent prepared using the method of any of embodiments 65-128 with one or more agents under conditions to reversibly bind the one or more agents to the particulate oligomeric reagents.

130. The method of Embodiment 127 or Embodiment 129, wherein the one or more agents comprise a binding partner, wherein the binding partner is capable of binding to one or more binding sites on the particulate oligomeric reagent.

131. The method of embodiment 130, wherein the binding partner comprises a streptavidin binding peptide.

132. The method of Embodiment 130 or Embodiment 131, wherein the binding partner comprises a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8 ), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser -His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln -Phe-Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

133. The method according to any one of embodiments 130-132, wherein one or more agents bind or are able to bind to a molecule expressed on the surface of the target cell.

134. The method according to any one of embodiments 130-133, wherein one or more agents comprises an antibody or an antigen-binding fragment thereof.

135. The method of embodiment 134, wherein the one or more agents is or contains a monovalent antibody fragment.

136. The method of Embodiment 134 or Embodiment 135, wherein one or more agents is or contains a Fab.

137. The method according to any one of embodiments 130-136, wherein the one or more agents is a receptor binding agent that binds or is capable of binding to a receptor expressed on the surface of a target cell.

138. The method according to embodiment 137, wherein the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, wherein the binding induces or modulates a signal in the target cell.

139. The method according to embodiment 137 or embodiment 138, wherein the target cell is an immune cell.

140. The method according to any one of embodiments 137-139, wherein the target cell is a T cell.

141. The method according to any one of embodiments 137-140, wherein the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3.

142. The method of Embodiment 141, wherein the stimulatory agent is the first receptor binding agent, and the method further comprises reversibly binding to an oligomeric particulate reagent of the second receptor binding agent, wherein the second receptor binding agent is capable of specifically binding to the second molecule on the surface of the target cell, and this binding to the second molecule is not necessarily capable of inducing or modulating a signal in the target cells.

143. The method of embodiment 142, wherein the second receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a TNF family member or TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains a molecule adhesion or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

144. The method of embodiment 142 or embodiment 143, wherein the second receptor binding agent specifically binds to a costimulatory molecule and the costimulatory molecule is CD28.

145. The method according to any of embodiments 127 and 129-144, wherein the one or more of the agents is an anti-CD3 antibody and an anti-CD28 antibody, optionally an anti-CD3 Fab and an anti-CD28 Fab.

146. The particulate oligomeric reagent of Embodiment 137 or Embodiment 138, wherein the receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a TNF family member, or a TNF family receptor, cytokine receptor, chemokine the receptor is or is or contains an adhesion molecule or a factor that causes cytokine production, chemokine production and/or expression of an adhesion molecule.

147. The method according to any one of embodiments 137, 138, 143, or 144, wherein the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1 ), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

148. The method according to any one of embodiments 137, 138, 143, or 144, wherein the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor and the cytokine receptor is selected from IL-2R, IL-1R, IL- 15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2.

149. The method according to any one of embodiments 137, 138, 143, or 144, wherein the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4.

150. The method according to any one of embodiments 137, 138, 143, or 144, wherein the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand that specifically binds to receptor for a cytokine or chemokine.

151. The method of Embodiment 150, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein

ligand specifically binds IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR type I, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2 ; and/or

the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or it is one of them biologically active fragment.

152. The particulate oligomeric reagent of Embodiment 151, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein the ligand specifically binds to a chemokine receptor selected from CCR1, CCR2 , CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or

the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof.

153. The method according to any one of embodiments 137, 138, 143, or 144, wherein the receptor binding agent (second receptor binding agent) is an adhesion molecule and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1 ), CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is their biologically active fragment.

154. The method of any one of embodiments 127-153, wherein the one or more agents is a selective agent, wherein the selective agent binds or is capable of binding to a selectable marker that is expressed on the surface of the target cell.

155. The method of embodiment 154, wherein the target cell is an immune cell.

157. The method according to embodiment 154 or embodiment 155, wherein the target cell is a lymphocyte or an antigen presenting cell.

158. The method according to any one of embodiments 154-156, wherein the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell.

159. The method according to any one of embodiments 154-158, wherein the target cell is a T cell.

160. The method according to any of embodiments 154-159, wherein the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

161. Composition containing particulate oligomeric reagents obtained using the method according to any embodiment 65-160.

162. A composition comprising a plurality of particulate oligomeric reactants obtained by the method of any embodiment 65-161.

163. A finished article containing a particulate oligomeric reagent according to any of embodiments 1-49 or a composition according to any of embodiments 50-64 or 161-162.

164. A method for modulating cells, the method comprising incubating a cell composition comprising target cells in the presence of a particulate oligomeric reagent according to any one of embodiments 1-49 or in the presence of a composition according to any one of embodiments 50-64 or 161-163, thus modulating target cells.

165. The method of embodiment 164 wherein modulating target cells comprises activating, enriching and/or expanding target cells.

166. A method for culturing cells, the method comprising incubating a cell composition comprising target cells in the presence of a particulate oligomeric reagent according to any one of embodiments 1-49 or in the presence of a composition according to any one of embodiments 50-64 or 161-163.

167. The method of any one of embodiments 164-166, wherein the particulate oligomeric reagent comprises a reversible bond with one or more agents.

168. The method of embodiment 167, wherein the one or more agents comprise a binding partner, wherein the binding partner is capable of binding to one or more binding sites on the particulate oligomeric reagent, thereby reversibly binding the one or more agents to the oligomeric reagent. in the form of particles.

169. The method of embodiment 168, wherein the binding partner comprises a streptavidin binding peptide.

170. The method according to embodiment 168 or 169, wherein the binding partner comprises a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His -Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe -Glu-Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

171. The method according to any one of embodiments 167-170, wherein one or more agents bind or are able to bind to a molecule expressed on the surface of the target cell.

172. The method according to any of embodiments 167-171, wherein one or more agents comprises an antibody, optionally a Fab.

173. The method according to any one of embodiments 167-172, wherein the one or more agents is a receptor binding agent that binds or is capable of binding to a receptor expressed on the surface of a target cell.

174. The method of Embodiment 173, wherein the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, thereby inducing or modulating a signal in the target cell.

175. The method according to embodiment 173 or embodiment 174, wherein the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3.

176. The method of embodiment 175, wherein the stimulatory agent is the first receptor binding agent, and the method further comprises reversibly binding to a particulate oligomeric reagent of the second receptor binding agent, wherein the second receptor binding agent is capable of specifically binding to the second molecule on the surface of the target cell, and this binding to the second molecule is not necessarily capable of inducing or modulating a signal in the target cells.

177. The method of embodiment 176, wherein the second receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a TNF family member or TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains a molecule adhesion or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

178. The method of embodiment 176 or 177, wherein the receptor binding agent specifically binds to a costimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a member of the TNF family or a TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains an adhesion molecule; or a factor that causes cytokine production, chemokine production, and/or expression of an adhesion molecule.

179. The method according to any one of embodiments 174, 175, 177, or 178, wherein the receptor binding agent (second receptor binding agent) binds to a costimulatory or accessory molecule and the costimulatory or accessory molecule is selected from CD28, CD90 (Thy-1 ), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM.

180. The method according to any one of embodiments 174, 175, 177, or 178, wherein the receptor binding agent (second receptor binding agent) specifically binds to a cytokine receptor and the cytokine receptor is selected from IL-2R, IL-1R, IL- 15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, type I IFNR, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2.

181. The method according to any of embodiments 174, 175, 177, or 178, wherein the receptor binding agent (second receptor binding agent) specifically binds to a chemokine receptor and the chemokine receptor is selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4.

182. The method according to any one of embodiments 174, 175, 177, or 178, wherein the receptor binding agent (second receptor binding agent) is a factor that induces the production of a cytokine or chemokine, and the factor is a ligand that specifically binds to receptor for a cytokine or chemokine.

183. The method of Embodiment 182, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a cytokine receptor, wherein

ligand specifically binds IL-2R, IL-1R, IL-15R, IFN-gammaR, TNF-alphaR, IL-4R, IL-10R, IFNR type I, IL-12R, IL-15R, IL-17R, TNFR1 and TNFR2 ; and/or

the ligand is selected from IL-2, IL-1, IL-15, IFN-gamma, TNF-alpha, IL-4, IL-10, IL-12, IL-15, IL-17 and TNF, or it is one of them biologically active fragment.

184. The method of Embodiment 183, wherein the receptor binding agent (second receptor binding agent) is a ligand that specifically binds to a chemokine receptor, wherein

the ligand binds specifically to a chemokine receptor selected from CCR1, CCR2, CCR3, CCR4, CCR5, CCR7, CCR9, CXCR1, CXCR3 and CXCR4; or

the ligand is selected from CXCL9, CXCL10, CCL19, CCL21 and CCL25, or it is a biologically active fragment thereof.

185. The method according to any one of embodiments 174, 175, 177, or 178, wherein the receptor binding agent (second receptor binding agent) is an adhesion molecule and the adhesion molecule is selected from CD44, CD31, CD18/CD11a (LFA-1 ), CD29, CD54 (ICAM-1), CD62L (L-selectin) and CD29/CD49d (VLA-4), CD106 (VCAM-1), or it is their biologically active fragment.

186. The method of any one of embodiments 164-185, wherein the one or more agents is a selective agent, wherein the selective agent binds or is capable of binding to a selectable marker that is expressed on the surface of the target cell.

187. The method of embodiment 186, wherein the target cell is an immune cell.

188. The method according to embodiment 186 or embodiment 187, wherein the target cell is a lymphocyte or an antigen presenting cell.

189. The method according to any one of embodiments 186-188, wherein the target cell is a T cell, B cell, NK cell, macrophage, or dendritic cell.

190. The method according to any one of embodiments 186-189, wherein the target cell is a T cell.

191. The method according to any of embodiments 186-190, wherein the selectable marker is CD25, CD28, CD62L, CCR7, CD27, CD127, CD3, CD4, CD8, CD45RA and/or CD45RO.

192. The method according to any one of embodiments 186-191, wherein the target cells include blood cells, leukocytes, lymphocytes, B cells, B cell population, T cells, T cell population, NK cells, dendritic cells and/or macrophages.

193. The method according to any one of embodiments 186-192, wherein the target cells express the recombinant receptor.

194. The method according to any one of embodiments 186-193, wherein the target cells express a recombinant T cell receptor and/or a chimeric antigen receptor (CAR).

195. The method according to any one of embodiments 186-194, wherein the target cells express a CAR that binds to an antigen associated with a disease and/or cancer.

196. The method of embodiment 195, wherein the antigen is αvβ6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), testicular cancer antigen, testicular cancer antigen 1B (CTAG, also known as NY-ESO-1 and LAGE-2), cancer embryonic antigen (CEA), cyclin, cyclin A2, C-C motif chemokine ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD138, CD171, epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), epidermal growth factor receptor type III mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrin-B2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor-like protein 5 (FCRL5; also known as Fc receptor homologue 5 or FCRH5), embryonic acetylcholine receptor (embryonic AchR), folate binding protein (FBP ), folate receptor alpha, fetal acetylcholine receptor, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gp100), Her2/neu (erbB2 receptor tyrosine kinase), Her3 (erb-B3), Her4 (erb -B4), erbB dimers, high molecular weight human melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, human leukocyte antigen A1 (HLA-AI), human leukocyte antigen A2 (HLA-A2), alpha- IL-22 receptor (IL-22Ra), IL-13 alpha receptor 2 (IL-13Ra2), kinase insert domain containing receptor (kdr), kappa light chain, L1 cell adhesion molecule (L1CAM), CE7 epitope L1-CAM leucine-rich repeat family A member 8 (LRRC8A), Lewis Y, melanoma-associated antigen (MAGE)-A1, MAGE-A3, MAGE-A6, mesothelin, c-Met, mouse cytomegavirus (CMV), mucin 1 (MUC1 ), MUC16, ligands of natural killer group D 2 members (NKG2D), melan A (MART-1), neural cell adhesion molecule (NCAM), oncoembryo flax antigen, preferably expressed melanoma antigen (PRAME), progesterone receptor, prostate-specific antigen, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), receptor tyrosine kinase-like orphan receptor 1 (ROR1), survivin, trophoblastic glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor gene (WT-1), or pathogen-specific antigen .

197. The method according to any one of embodiments 186-196, further comprising breaking the reversible bond between one or more agents and the particulate oligomeric reagent.

198. The method of embodiment 197, wherein said disruption comprises administering to the target cells a composition containing a substance capable of flipping the bond between one or more agents and the particulate oligomeric reagent.

199. The method of embodiment 198, wherein the agent is a free binding partner and/or a competitive agent.

200. The method according to any one of embodiments 197-199, wherein said gap terminates or attenuates a signal induced or modulated by one or more agents in target cells, optionally T cells.

201. The method according to any one of embodiments 197-200, wherein the substance comprises a streptavidin binding peptide, biotin or biologically active fragment, optionally D-biotin or biotin analogue or biologically active fragment.

202. The method of Embodiment 201, wherein the substance is a streptavidin binding peptide selected from the group consisting of Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 8), Trp-Ser -His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₃ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys ((SEQ ID NO: 17), Trp-Ser-His-Pro- Gln-Phe-Glu-Lys-(GlyGlyGlySer) ₂ -Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 18) and Trp-Ser-His-Pro-Gln-Phe-Glu- Lys-(GlyGlyGlySer) ₂ Gly-Gly-Ser-Ala-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 19).

203. The method according to any one of embodiments 197-202, wherein the break is carried out within 5 days after the start of said incubation.

204. The method of any one of embodiments 164-203, wherein the one or more receptor binding agents comprise an antibody or antigen-binding fragment thereof.

205. The method of embodiment 204, wherein the one or more receptor binding agents is or contains a monovalent antibody fragment.

206. The method according to embodiment 204 or embodiment 205, wherein one or more agents is or contains a Fab.

207. The method according to any one of embodiments 164-206, wherein the target cell is an immune cell.

208. The method according to any one of embodiments 164-207, wherein the target cell is a T cell.

209. The method of any one of embodiments 176-208, wherein the receptor binding agent is or contains a stimulatory agent capable of binding to a molecule on the surface of a target cell, wherein the binding induces or modulates a signal in the target cell.

210. The method according to any one of paragraphs. 176-209, wherein the receptor-binding agent is capable of initiating a TCR/CD3 complex-associated signal in T cells, binds to an element of the TCR/CD3 complex; and/or specifically binds to CD3.

211. The method according to p. 209 or p. 210, wherein the stimulating agent is the first receptor-binding agent, and the oligomeric particulate reagent contains a second receptor-binding agent, while the second receptor-binding agent is able to specifically bind to the second molecule on the surface of the target cell, wherein binding to the second molecule is optionally capable of inducing or modulating a signal in the target cells.

212. The method of claim 211, wherein the second receptor-binding agent specifically binds to a co-stimulatory molecule, an accessory molecule, an immune checkpoint molecule, is a TNF family member or TNF family receptor, a cytokine receptor, a chemokine receptor, or is or contains a molecule adhesion or a factor that causes the production of a cytokine, the production of a chemokine and/or the expression of an adhesion molecule.

213. The method of claim 211 or claim 212, wherein the second receptor-binding agent specifically binds to a costimulatory molecule and the costimulatory molecule is CD28.

214. The method according to any one of paragraphs. 176-213, wherein one or more of the agents is an anti-CD3 antibody and an anti-CD28 antibody, optionally an anti-CD3 Fab and an anti-CD28 Fab.

VIII. Examples

The following examples are included for illustrative purposes only and are intended to limit the scope of the invention.

Example 1: Methods for preparing an oligomeric reagent containing a streptavidin mutein.

The oligomeric reagent was prepared by polymerizing an exemplary streptavidin mutein designated STREP-TACTIN® M2 (a streptavidin homotetramer containing the amino acid sequence of the mutein set forth in SEQ ID NO:61, see e.g. US Pat. No. 6,103,493 and Voss and Skerra (1997) Protein Eng 1:975-982). To prepare streptavidin muteins for oligomerization, streptavidin muteins containing one or more reactive thiol groups were incubated with maleimide-activated streptavidin muteins. To prepare streptavidin mutein, approximately 100 mg of streptavidin mutein tetramer was thiolated by incubation with 2-iminothiolane hydrochloride at a molar ratio of 1:100 at a pH of approximately 8.5 at 24°C for 1 hour in 100 mM borate buffer in a total volume of 2.6 ml . For the maleimide activation reaction, approximately 400 mg of streptavidin mutein tetramer was incubated with succinimidyl-6-[(β-maleimidopropionamido)hexanoate (SMPH) at a molar ratio of 1:2 at a pH of approximately 7.2 at 24°C for 1 hour in total volume of approximately 10.4 ml in sodium phosphate buffer. The thiolation and maleimide activation reactions were coordinated so that they started at approximately the same time and the duration of the reactions was controlled.

After the reactions, 2-iminothiolane hydrochloride and SMPH that did not react with STREP-TACTIN® M2, along with low molecular weight reaction by-products (predominantly NHS), were removed from the samples by gel filtration of the samples individually using PD-10 desalting columns (GE healthcare). For each 2.5 ml sample, a PD-10 column (8.3 ml bed volume) was equilibrated and loaded with either thiolated streptavidin mutein or streptavidin maleimide mutein and eluted by adding 3.5 ml of binding buffer (100 mM NaH ₂ P ₄ , 150 mM NaCl, 5 mM EDTA, pH 7.2). Streptavidin maleimide-mutein gel filtration was performed on 4 columns with a volume of >10 ml, and the eluates were pooled. The time of the activation and thiolation reactions and the time between the end of the activation and thiolation reactions and the start of the oligomerization reactions were carefully controlled. Typically, after or about ten minutes from the beginning of the gel filtration, that is, at the end of the activation and thiolation reactions, the oligomerization reaction was started.

For oligomerization, samples of streptavidin maleimide mutein and thiolated streptavidin mutein were then combined into a total volume of approximately 17.5 ml and incubated for 1 hour at pH 7.2 at 24° C. under stirring conditions at about 600 rpm. Since four times more streptavidin mutein was incubated with SMPH than with 2-iminothiolane hydrochloride, the molar ratio of thiolated streptavidin mutein to maleimide-streptavidin mutein during the oligomerization reaction was 1:4. After the reaction, the remaining SH groups of the oligomerized streptavidin mutein reagent were saturated by incubation with N-ethylmaleimide (NEM) for 15 minutes at 24° C. with stirring (about 600 rpm), followed by incubation for another 16-20 hours at 4° FROM.

After incubation with NEM, the sample containing the oligomerized Strep-Tactin mutein was centrifuged and the supernatant filtered through a 0.45 µm membrane (Millex-HP 0.45 µm from Merck Millipore). The filtered solution was then loaded onto a size exclusion chromatography (SEC) column (Sephacryl S-300 HR HiPrep 26/60, GE Healthcare) using an AKTA Explorer chromatography system (GE Healthcare). Fractions having at the end of collection milli-units of optical density (mAU) greater than or equal to 1500 mAU were pooled.

The pooled sample containing streptavidin oligomeric mutein was treated with 100 mM hydroxylamine by adding 890 mM pH 6.35 over 15 minutes at room temperature. To remove hydroxylamine after treatment, the sample was loaded onto a PD10 column (2.5 ml per column) equilibrated with 100 mM NaH ₂ PO ₄ , 140 mM NaCl, 1 mM EDTA, pH 7.2, and eluted with 3.5 ml of the same buffer (100 mM NaH ₂ PO ₄ , 140 mM NaCl, 1 mM EDTA, pH 7.2.) PD10 eluates were pooled and sterile filtered through a 0.45 µm filter followed by a 0.22 µm filter, and then the samples were frozen and stored at - 80°C. Before freezing, the final concentration of the streptavidin mutein oligomeric reagent was measured, and the size of the streptavidin oligomeric mutein reagent was determined by dynamic light scattering (DLS).

To evaluate the sequence of the oligomerization process, 10 streptavidin mutein oligomeric reagents were prepared using the methods described above from five different batches of streptavidin mutein (SAM). the average size, percentage yield (determined by measuring absorbance at 280 nm without base correction) and activity (biotin binding) of the oligomers were evaluated, and the results are shown in Table E1. The results showed that the resulting streptavidin mutein oligomeric reagents were consistent in these parameters with an average radius of 97 nm ± 10 nm and a biotin binding of 40 nmol/mg ± 3 nmol/mg.

Table E1: Comparison of oligomerized STREP-TACTIN from different batches.

SAM series Radius (nm) Exit (%) Binding of Biotin
(nmol/mg) Party 1 one 92 74 41 Party 2 2 100 68 40 Party 3 2 106 82 37 Party 4 2 94 73 39 Party 5 3 87 79 41 Party 6 3 90 81 39 Party 7 four 97 84 43 Party 8 four 97 76 43 Party 9 5 102 85 42 Party 10 5 87 63 42

The average molecular weight (MW) of the three oligomeric streptavidin mutein reagents prepared as described above was measured by asymmetric in-field flow fractionation (AF4) performed with an HPLC system (AGILENT 1100 and Wyatt ECLIPSE DUALTEC) with UV detection ( Agilent UV detector in combination with MALLS DAWN HELEOS (Wyatt)). AF4 measurements allowed the calculation of the average number of streptavidin mutein tetramers in each oligomeric reagent, assuming an average molecular weight of streptavidin mutein tetramer of 52,500 g/mol (52.5 kDa) (Table E2).

Table E2: Size and Molecular Weight of Oligomeric Streptavidin Mutein Reagents

Radius (nm) MW (g/mol) Number of tetramers 102 1.65×10 ⁸ 3150 82 ^1.08x108 2050 92 ^1.26x108 2280

Example 2 Evaluation of parameters affecting the oligomerization of the streptavidin mutein reagent.

Evaluated various parameters in the method described in example 1, to assess the impact on various aspects of the method of creating oligomeric reagent mutein streptavidin.

A. pH level

The effect of carrying out the thiolation reaction at various pH values on the oligomer size and product yield was evaluated. Iminothiolane was purchased as the hydrochloride and its dissolution in 100 mM borate buffer at pH 8.5 to a concentration of 100 mg/ml caused a pH drop of ± 0.7 units to pH 7.8. When the borate buffer was used at a lower strength, ie 25 mM, the addition of iminothiolane hydrochloride to a concentration of 100 mg/ml resulted in a larger drop in pH from 1.6 units to pH 6.9. Thiolation reactions were performed at various pH values by incubating 100 mg of streptavidin mutein with 2-iminothiolane hydrochloride in a molar ratio of 1:100 in 25 mM borate buffer at pH 7.5, pH 8.5 and pH 9.5. The thiolyzed streptavidin mutein was combined with the maleimide-activated streptavidin mutein and the oligomerization process was carried out essentially as described in Example 1. As shown in Table E3, lowering the pH of the borate buffer used in the thiolation reaction resulted in a decrease in oligomer size and yield, while an increase in pH resulted in an increase in size and yield. An inadvertent loss of material occurred during the experiment at pH 9.5, resulting in a decrease in yield. The value in brackets in Table E3 reflects the calculated output in the absence of losses.

Table E3: Size and Yield of STREP-TACTB Oligomer after Thiolation at Different pH

pH Thiolation Reactions Radius (nm) Exit (%) pH 7.5 fourteen 6 pH 8.5 40 48 pH 9.5 52 14 (62)

To evaluate whether a stronger buffer (i.e., 100 mM borate buffer versus 25 mM with a concomitant increase in pH in the final reaction mixture) changes the reaction kinetics, the iminothiolane activation reaction was performed as described in Example 1. net thiol functionality was determined using Elman's reagent at several time points during the reaction. The reaction rate in 100 mM borate buffer is shown in FIG. 1 and was found to be faster and introduce more SH groups than when the reaction was run in 25 mM borate buffer. The iminothiolane activation reaction reached a peak concentration of thiol functional groups starting as early as 25 minutes after the start of the reaction in 100 mM borate buffer, compared to 50-150 minutes when the reaction was carried out in 25 mM borate buffer, which is the result of a higher final pH, at where the reaction occurs when using 100 mM buffer instead of 25 mM buffer.

The effect of pH on other stages of the oligomerization process was also evaluated. Oligomerization reactions were carried out with reaction buffers (25 mM borate) at various pH values, 8.3 and 8.5, and with binding buffers (100 mM phosphate) at various pH values, 7.0 and 7.2. Performing the thiolation reaction at a lower pH (8.3 instead of 8.5) had a greater effect on oligomer size than performing the activation or coupling reactions at a lower pH (7.0 instead of 7.3). However, carrying out the activation reaction or coupling reaction at pH 7.0 did reduce the size of the oligomer compared to carrying out the reactions at pH 7.2.

B. Effect of Timing and pH on the Thiolation Reaction

Iminothiolane-activated (thiolated) streptavidin mutein analysis was performed to determine whether degradation of the thiol functionality was due to disulfide formation (which cannot react with maleimide) or isomerization towards the N-substituted iminothiolane.

In one experiment, the net content of thiol functional groups was determined at different time points using Elman's reagent by measuring the absorbance at 412 nm after the thiolation reaction with iminothiolane. The reaction was carried out with 25 mM borate buffer, which had an initial pH of 8.3 or 8.5, although the actual pH during the reaction was slightly below 7. As shown in FIG. 2, the thiol functionality peaked between 50 and 150 minutes (earlier with pH 8.5 and later with pH 8.3) and the height was pH dependent (higher with pH 8.5 compared with pH 8 ,3). The content of thiol functional groups in the streptavidin mutein during the reaction at both pH values converged after 3 hours, demonstrating that after 3 hours the streptavidin mutein prepared using the higher pH of 8.5 contained more N-substituted iminothiolane. Additional experiments showed that the reduction in SH function was not affected by the addition of the reducing agent tris(2-carboxyethyl)phosphine (TCEP), which would reverse direction if the cause of the reduction in SH function was disulfide formation.

In addition, streptavidin muteins were thiolated by incubation with iminothiolane for 10 minutes, 50 minutes, or 390 minutes using 25 mM borate buffer at pH 8.3 and 8.5, and the reaction products were analyzed by size exclusion chromatography (SEC) using speed flow 0.4 ml/min with 100 mm NaH ₂ PO ₄ , 140 mm NaCl, 1 mm EDTA, pH 7.0 and using an Agilent Bio SEC-3 column 3 μm 300Å. Molecular weight standards (indicated by peaks at 158,000 Da, 44,000 Da, 17,000 Da, or 1350 Da in Figure 3) were used for size determination. The SEC analysis is shown in FIG. 3, in which the unactivated streptavidin mutein tetramer is shown in dotted line. The unactivated mutein tetramer was found to have the lowest retention time, while thiolated samples migrate more slowly (corresponding to lower molecular weight) the longer they react with iminothiolane. The observed peaks of the samples that reacted with iminothiolane were essentially identical in size and no peaks with lower retention times were observed. Thus, it can be concluded from the analysis that higher-order dimers or oligomers are not formed in the long-term reaction with iminothiolane, which should have taken place in the case of the formation of disulfide.

To assess the effect of time on SH reduction, the net thiol functionality at the end of 1-h or 3-h activation of streptavidin mutein iminothiolane at various pH values (pH 8.3, 8.5, or 8.7) was evaluated in 25 mM borate buffer. After the reaction and PD10 gel filtration, the SH content was determined by measuring the absorbance at 412 nM after the addition of Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoate) (DTNB). As a result of the reaction, 5-thio-2-nitrobenzoate ion TNB2- is released, which has an absorption maximum at 412 nm. By measuring the increase in absorbance at 412 nm and dividing by the molar extinction coefficient of the TNB2- ion at 412 nm, the free sulfhydryl content of the molecule can be calculated. As shown in FIG. 4, while the content of thiol functional groups after a 1 hour reaction depended on pH, after a 3 hour reaction, very little effect of pH on the content of thiol functional groups was evident. It is hypothesized that a longer activation reaction may reduce the effect of pH on thiol functionality and thus make the reaction more robust against pH dependent size changes, but may also increase N-substituted iminothiolane, which may be a source of long term size instability. due to repeated isomerization.

In order to measure the reduction in thiol functional groups in the absence of thiol formation, the content of thiol functional groups was measured at several time points after the end of the thiolation reaction, that is, after removal of the iminothiolane reagent. The SH content of thiolated streptavidin was measured with Ellman's reagent at various times after removal of 2-iminothiolane by PD10 gel filtration. As shown in FIG. 5, 5 minutes after the end of the reaction, 5% destruction of the thiol functional groups was detected, 60 minutes after the reaction, 26% destruction was detected, and 45% destruction of the thiol functional groups was detected 120 minutes after the reaction. The half-SH loss period was calculated to be 139 min. This shows the result of using a different time frame between the end of iminothiolane and SMPH activation and the start of the multimerization reaction.

C. Molar ratios of streptavidin mutein and SMPH

To assess the effect of the concentration of SMPH and streptavidin mutein in the SMPH activation reaction on the oligomerization of the streptavidin mutein, oligomerization procedures were performed with various concentrations (within ± 10%) of SMPH and streptavidin mutein. An increase or decrease in the concentration of SMPH or streptavidin by 10% resulted in marked differences in the size of the streptavidin mutein oligomer. In some aspects, it is recommended that the accuracy of these parameters (Strep-Tactin and SMPH concentration) be better than ±2%.

D. Oligomer Stabilization

As a result of thiolation, the unreacted N-substituted iminothiolane can remain on the lysine residues and on the free N-terminus of the streptavidin mutein and contribute to the postsynthetic increase in the oligomer size. To evaluate the effect of hydroxylamine treatment on postsynthetic oligomer growth, oligomerization procedures were performed as described in Example 1, except that the procedures included either a 1 hour activation reaction with iminothiolane (samples 1 and 2) or a 3.5 hour activation reaction. with iminothiolane (samples 3 and 4). In addition, in these studies, after SEC, samples containing oligomeric streptavidin mutein reagents were either treated with 100 mM hydroxylamine (HA) at pH 6.35 for 15 minutes at room temperature (samples 1 and 3) or not treated with hydroxylamine (samples 2 and 4). The samples were divided into fractions, which were stored at -80°C, 4°C or 37°C. Oligomer size was measured by DLS immediately after synthesis (day 0) and 1 week, 3 weeks and 9 weeks after synthesis. The results are shown in Table E4.

Table E4: Effect of Storage Temperature and Hydroxylamine Treatment on Postsynthetic Growth

Sample Storage conditions Radius on d0 Radius after w1 Radius after w3 Radius after w9 Radius after w27 sample 1
+HA 1 h -80°C 89 nm 94 nm 93 nm 91 nm 91 nm 4°C 89 nm 95 nm 95 nm 96 nm 100 nm 37°C 89 nm 99 nm 108 nm 107 nm 114 nm Sample 2
-HA 1 h -80°C 88 nm 93 nm 96 nm 94 nm 94 nm 4°C 88 nm 97 nm 106 nm 106 nm 112 nm 37°C 88 nm 115 nm 137 nm 178 nm 329 nm Sample 3
+HA 3.5 h -80°C 94 nm 100 nm 98 nm 94 nm 92 nm 4°C 94 nm 96 nm 98 nm 100 nm 102 nm 37°C 94 nm 108 nm 121 nm 142 nm 249 nm Sample 4
-HA 3.5 h -80°C 91 nm 98 nm 98 nm 96 nm 100 nm 4°C 91 nm 111 nm 138 nm 157 nm 99 nm 37°C 91 nm 148 nm 177 nm 265 nm 409 nm

As shown in Table E4, hydroxylamine treatment reduces oligomer growth during storage at 4°C and 37°C. It was observed that when stored at -80°C, the size remains stable regardless of the treatment with hydroxylamine. Samples 3 and 4 were activated for 3.5 hours and showed more pronounced postsynthetic growth of oligomers. Without wishing to be limited by theory, in some aspects, an activation time of 3.5 hours is not as sensitive to changes in pH as an activation time of 1 hour (see Example 2B), but may result in higher levels of N-substituted iminothiolane. Together, the results showed that hydroxylamine treatment reduced post-synthetic growth of streptavidin mutein oligomers. The data also demonstrated that storage at -80°C improves the stability of the oligomers. Thus, in some aspects, combining both treatments with hydroxylamine followed by storage at -80° C. may provide improved stability for maintaining a constant size, including long-term storage.

Example: Creation of a soluble stimulatory reagent containing oligomerized anti-CD3 and anti-CD28 Fab fragments reversibly linked to streptavidin mutein oligomers.

The stimulatory agents (anti-CD3 and anti-CD28 Fab fragments) were multimerized by reversibly binding to the streptavidin mutein oligomeric reagent prepared as described in Example 1. The anti-CD3 and anti-CD28 Fab fragments were reversibly bound to the streptavidin oligomeric mutein via the streptavidin peptide-binding partner. , merged with each Fab fragment. The anti-CD3 Fab was derived from a CD3-binding monoclonal antibody produced by the OKT3 hybridoma cell line (ATCC® CRL-8001™; see also US Pat. No. 4,362,549) and contained the heavy chain variable domain and the light chain variable domain of the OKT3 anti-CD3 antibody. described in Arakawa et al., J. Biochem. 120, 657-662 (1996). These sequences are shown in SEQ ID NOs: 31 and 32, respectively. An anti-CD28 Fab was generated from the CD28.3 antibody (deposited as a synthetic single chain Fv construct under GenBank accession number AF451974.1; see also Vanhove et al., BLOOD, July 15, 2003, vol. 102, no. 2, pages 564-570) and contained the heavy and light chain variable domains of the CD28.3 anti-CD28 antibody shown in SEQ ID NOS: 33 and 34, respectively. The Fab fragments were individually fused at the carboxy-terminus of their heavy chain to a streptavidin peptide binding sequence containing a serial arrangement of two streptavidin binding modules having the amino acid sequence SAWSHPQFEK (GGGS) 2GGSAWSHPQFEK (SEQ ID NO: 16). Peptide-labeled Fab fragments were generated recombinantly (see International Patent Application Publication No. WO 2013/011011 and WO 2013/124474).

To effect reversible binding, peptide-labeled anti-CD3 and anti-CD28 Fab fragments were mixed with the streptavidin oligomeric mutein reagent at approximately room temperature, thereby reversibly binding them to the reagent via interaction between twin-strep tags on the Fab fragments, which were binding partners capable of reversibly bind to binding sites on the reagent. In some cases, peptide-labeled Fab fragments were premixed prior to immobilization on the streptavidin mutein oligomeric reagent, which in some cases may result in a more uniform distribution of the various Fab molecules.

Example 4 Evaluation of the Anti-CD3 and Anti-CD28 Activity of Oligomerized Fab Fragments Reversibly Linked to Streptavidin Mutein Oligomers

Anti-CD3 and anti-CD28 Fab fragments reversibly associated with various streptavidin oligomeric reagents from each lot described in Table E1 were evaluated for T-cell stimulatory capacity using the process described in Example 1. These oligomeric streptavidin reagents had an average radius of about 95 nm. The metabolic activity of cells as an indicator of cell proliferation was assessed by colorimetric monitoring of the cleavage of the stable tetrazolium salt WST-1 to a soluble dye-formazan complex.

T cells from three different donors were incubated with multimerized anti-CD3/anti-CD28 Fab fragments reversibly linked to the streptavidin oligomeric reagent. Cells were also incubated with control oligomeric reagents that had an average radius of 101 (internal standard) or 36 nm, which were also reversibly linked to anti-CD3/anti-CD28 Fab fragments. After incubation, the WST-1 reagent was applied to the cells and metabolic activity levels were assessed by measuring absorbance at 450 nm as an indication. The results were normalized by the number of cells in the analyzed culture and are presented as the ratio of WST-1 to the number of cells.

As shown in FIG. 6B, the mean activity of WST-1 T cells stimulated with each of the tested reagents was comparable. In addition, the degree of stimulation was the same for all tested reagents and was comparable to the same size of the internal reference reagent (usually varied within ± 2 standard deviations). On figa shows the activity of WST-1, depicted as a separate data point for each reagent. In Fig. 6A and Fig. 6B shows that T cell stimulation, as observed by WST-1 activity, was lower with anti-CD3/anti-CD28 Fabs multimerized on the smaller 36 nm backbone of the streptavidin mutein oligomeric reagent.

Example 5: An illustrative method for the preparation of an oligomeric streptavidin mutein.

Streptavidin mutein oligomeric reagents were prepared using an illustrative method similar to that described in Example 1 with some modifications. Specifically, after incubation with NEM, the sample containing the oligomerized streptavidin mutein was centrifuged, and the supernatant was filtered through a 0.45 μm membrane. The filtered solution was then treated with 100 mM hydroxylamine at pH 6.35 for 15 minutes at room temperature. The sample was then loaded onto a size exclusion chromatography (SEC) column using an AKTA Explorer chromatography system (GE Healthcare). Fractions with milli-unit absorbance (mAU) greater than or equal to 1500 mAU were pooled and then sterile filtered through a 0.45 µm filter and then a 0.22 µm filter. The oligomerized streptavidin was stored at -80°C. Thus, in contrast to the method described in Example 1, no additional gel filtration based on PD-10 was performed.

The size and stability of the streptavidin mutein oligomeric reagents prepared according to the illustrative method were compared with those prepared according to the method described in Example 1. The oligomers were separated into fractions, which were stored at -80°C, 4°C or 37°C. The size of the oligomer was measured by DLS immediately after synthesis (day 0) and at various time points after synthesis. In both methods, regardless of whether hydroxylamine was removed after SEC or before SEC, oligomers of comparable size and stability were obtained. As shown in FIG. 11, oligomers stored at -80°C or 4°C showed an average size change of less than 10% over 46 weeks after synthesis. Oligomers stored at 37°C showed an increase in size of about 50% after 9 weeks of synthesis.

Example 6: Evaluation of different batches of streptavidin mutein oligomeric reagents prepared

Different batches of oligomeric streptavidin mutein reagents, consisting of different batches of starting material, were evaluated as part of the chimeric antigen receptor (CAR) T cell construction process. Three separate batches of oligomeric streptavidin mutein reagents were prepared from separate batches of STREP-TACTIN® M2, essentially as described above. Anti-CD3 and anti-CD28 Fabs containing a streptavidin peptide binding fusion partner from the same production lot were reversibly linked to separate streptavidin mutein oligomers from each lot, essentially as described above. Three separate batches of oligomeric streptavidin mutein reagents had average diameters from 100 to 109 nm.

Three separate series of oligomeric streptavidin mutein reagents conjugated to anti-CD3/anti-CD28 Fab were used to stimulate primary CD4+ and CD8+ T cells. CD4+ and CD8+ T cells were individually selected from an apheresis sample from three separate donors prior to pooling the selected cells at a defined ratio of CD4+ T cells to CD8+. CD4+ and CD8+ combined T cells were then treated with a chimeric antigen receptor (CAR) using an exemplary design method that involved incubating the cells with a large amount of streptavidin mutein oligomeric reagents conjugated to anti-CD3/anti-CD28 Fab, transduced with lentivirus, and then growing for reproduction. Before completion of expansion, the cells were treated with biotin to dissociate the anti-CD3/anti-CD28 Fab from the streptavidin oligomeric reagents. Conditions for incubation, transduction, and culture were the same for all batches of anti-CD3/anti-CD28 Fab-conjugated streptavidin mutein oligomeric reagents tested, and each batch was tested using up to four replicates for each of three donor samples. To control the transduction efficiency of the engineered T cells, the lentivirus delivered a polynucleotide encoding CAR, separated from the truncated receptor by a ribosome slip sequence, to be used as a surrogate marker to detect CAR expression.

the total number of viable cells was quantified at various stages of the exemplary process. Prior to addition of the Fab-conjugated streptavidin mutein oligomeric reagent (day 0) and on different days of the process, the cells were stained for viability. As shown in FIG. 12A and 12B, the total viable cell count and percentage of viable cells measured at different stages of the construction process were the same for cells obtained from different donors and using different lots of Fab-conjugated streptavidin mutein oligomeric reagents.

Transduction efficiency was measured in cells harvested from the exemplary design method. Cells were labeled with antibodies specific for CD4, CD8 and a surrogate marker and analyzed by flow cytometry. The percentage of CAR+, CAR+ CD4+, CAR+ CD8+ cells among all viable T cells is shown in FIG. 13. Transduction efficiency was similar among the various Fab-conjugated streptavidin mutein oligomeric reagents, and the observed variability was mainly dependent on the donor source.

Engineered CAR+ T cells were also tested for various markers associated with memory or exhaustion phenotypes. T cells were labeled with antibodies specific for CD3, CD4, CD8 and surrogate markers, as well as markers associated with memory CCR7, CD27, CD28, CD45RA, CD45RO and CD62L, or markers associated with depletion of CCR7, LAG3, PD-1 and TIM3 and then analyzed by flow cytometry. There were no significant changes in staining for markers associated with memory or exhaustion. A principal component analysis was performed for phenotypic markers associated with memory and exhaustion. Clusters derived from comparisons of cumulative data, separated by donor rather than individual series of Fab-conjugated streptavidin mutein oligomeric reagents, are consistent with the composition of the reagents for memory- and depletion-associated T cell phenotypes that may occur during the construction process.

Engineered CAR+ T cells were analyzed for residual Fab or streptavidin mutein on cell surfaces by detecting residual anti-CD3 and anti-CD28 Fabs by StrepTactin-PE staining or by detecting residual streptavidin mutein with an antibody specific for StrepTactin. As shown in FIG. 14, less than 1% of viable T cells were positive for Fab or streptavidin mutein.

CAR-dependent antigen activity was assessed by co-culturing engineered CAR+ T cells with irradiated cells expressing the CAR target antigen. T cells cultured in the absence of irradiated antigen-expressing cells served as a negative control. After four hours of co-culture, T cells were assessed for intracellular IL-2, IFN-gamma, and TNF-alpha by intracellular cytokine staining (ICS) stained for a surrogate marker indicative of CAR and CD3 expression and assessed by flow cytometry. Similar percentages of cells positive for IL-2, IFN-gamma, and TNF-alpha, as well as percentages of cells expressing two or three cytokines, were observed among CAR+ CD3+ T cells derived from different donors and among cells produced in the presence of different series of Fab-conjugated oligomeric streptavidin mutein reagents.

Cytolytic activity was assessed in engineered CAR+ T cells after co-culture of the cells with target cells expressing the CAR target antigen at a ratio of effector cells to target cells of 5:1. Adhering target cells were seeded in the wells of an electronic microtiter plate, and the number of target cells was quantified by measuring the electric current impedance between the attached electrodes. As a control, target cells were cultured alone or with T cells that were incubated during an illustrative design process with a paramagnetic bead reagent conjugated to reference anti-CD3/anti-CD28- antibodies. As shown in FIG. 15, CAR+ T cells in the assay showed efficient killing. The cytolytic activity observed was comparable among T cells obtained from different individual donors and among different batches of Fab-conjugated streptavidin mutein oligomeric reagents obtained.

Taken together, these data are highly consistent with the composition of the Fab-conjugated oligomeric streptavidin mutein reagents prepared using the methods provided.

The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications of the described compositions and methods will become apparent from the description and ideas given here. Such variations may be practiced without departing from the true scope and spirit of the disclosure, and are intended to fall within the scope of the present disclosure.

SEQUENCES

No. Subsequence Description one DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ Streptavidin
Species: Streptomyces avidinii
UniProt No. P22629 2 EAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS minimal streptavidin
Species: Streptomyces avidinii 3 DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ Streptavidin mutein Val44-Thr45-Ala46-Arg47
Species: Streptomyces avidinii four EAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS Mutein Streptavidin Val44-Thr45-Ala46-Arg47
Species: Streptomyces avidinii 5 DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYIGARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQ Mutein Streptavidin Ile44-Gly45-Ala-46-Arg47
Species: Streptomyces avidinii 6 EAGITGTWYNQLGSTFIVTAGADGALTGTYIGARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS Mutein Streptavidin Ile44-Gly45-Ala-46-Arg47
Species: Streptomyces avidinii 7 Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Streptavidin binding peptide, Strep-tag® eight WSHPQFEK Strep-tag® II 9 His-Pro-Baa Streptavidin-binding peptide
Baa is selected from glutamine, asparagine and methionine ten His-Pro-Gln-Phe Streptavidin-binding peptide eleven Oaa-Xaa-His-Pro-Gln-Phe-Yaa-Zaa Streptavidin-binding peptide
Oaa is Trp, Lys or Arg;
Xaa is any amino acid;
Yaa represents Gly or Glu
Zaa is Gly, Lys or Arg 12 -Trp-Xaa-His-Pro-Gln-Phe-Yaa-Zaa- Streptavidin-binding peptide
Xaa is any amino acid;
Yaa represents Gly or Glu
Zaa is Gly, Lys or Arg 13 Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(Xaa)n-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys- Sequential modules of streptavidin-binding peptide
Xaa is any amino acid;
n is either 8 or 12 fourteen Trp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)n-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys Sequential modules of streptavidin-binding peptide
n is 2 or 3 fifteen SAWSHPQFEKGGGSGGGSGGGSWSHPQFEK twin strep tag 16 SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK twin strep tag 17 WSHPQFEKGGGSGGGSGGGSWSHPQFEK twin strep tag eighteen WSHPQFEKGGGSGGGSWSHPQFEK twin strep tag 19 WSHPQFEKGGGSGGGSGGSAWSHPQFEK twin strep tag twenty Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala HA-tag 21 Tyr-Thr-Asp-Ile-Glu-Met-Asn-Arg-Leu-Gly-Lys VSV-G-tag 22 Gln-Pro-Glu-Leu-Ala-Pro-Glu-Asp-Pro-Glu-Asp HSV tag 23 Ala-Ser-Met-Thr-Gly-Gly-Gln-Gln-Met-Gly T7 epitope 24 Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu HSV epitope 25 Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu Myc-epitope 26 Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr V5-tag 27 EAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEENAGYSTLVGHDTFTKVKPSAAS Mutein Streptavidin Ile44-Gly45-Ala-46-Arg47 and Glu117, Gly120, Try121 (m1-9 mutein)
Species: Streptomyces avidinii 28 DPSKDSKAQVSAAEAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEENAGYSTLVGHDTFTKVKPSAAS Mutein Streptavidin Ile44-Gly45-Ala-46-Arg47 and Glu117, Gly120, Try121 (m1-9 mutein)
Species: Streptomyces avidinii 29 AMQVQLKQSGPGLVQPSQSLSITCTVSGFSLTTFGVHWVRQSPGKGLEWLGVIWASGITD YNVPFMSRLSITKDNSKSQVFFKLNSLQPD DTAIYYCAKNDPGTGFAYWGQGTLVTVSAGSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCGSAWSHPQ FEKGGGSGGG SGGSAWSHPQFEK Heavy chain variable domain of Fab fragment m13B8.2 thirty AMDIQMTQSPASLSASVGETVTFTCRASEMIYSYLAWYQQKQGKSPQLLVHDAKTLAEGVPSRFSGGGSGTQFSLKINTLQPEDFGTYYCQAHYGNPPTFGGGTKLEIKRGIAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAGLACEK Light chain variable domain of the m13B8.2 Fab fragment 31 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Heavy chain variable domain of OKT3 anti-CD3 antibody 32 Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Light chain variable domain of OKT3 anti-CD3 antibody 33 Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile Ile His Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly Trp Phe Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met Glu Leu Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg Asp Asp Phe Ser Gly Tyr Asp Ala Leu Pro Tyr Trp Gly Gln Gly Thr Met Val Thr Val Heavy chain variable domain of anti-CD28 antibody CD28.3 34 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly Glu Thr Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile Tyr Ser Asn Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Leu Gln Ser Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Cys Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Anti-CD28 antibody CD28.3 light chain variable domain 35 His-Asn-His-Arg-His-Lys-His-Gly-Gly-Gly-Cys MAT-tag 36 YNLDVRGARSFSPPRAGRHFGYRVLQVGNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPTDGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQECIKGNVDLVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFSDYVKRKDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIIITDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETLHKFASKPASEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGAKDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLASGAPRYQH MGRVLLFQEPQGGGHWSQVQTIHGTQIGSYFGGELCGVDVDQDGETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITALTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQWFGRSIHGVKDLEGDGLADVAVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVHEVECSYSTSNKMKEGVNITICFQIKSLIPQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTPRDQRAQGKDIPPILRPSLHSETWEIPFEKNCGEDKKCEANLRVSFSPARSRALRLTAFASLSVELSLSNLEEDAYWVQLDLHFPPGLSFRKVEMLKPHSQIPVSCEELPEESRLLSRALSCNVSSPIFKAGHSVALQ MMFNTLVNSSWGDSVELHANVTCNNEDSDLLEDNSATTIIPILYPINILIQDQEDSTLYVSFTPKGPKIHQVKHMYQVRIQPSIHDHNIPTLEAVVGVPQPPSEGPITHQWSVQMEPPVPCHYEDLERLPDAAEPCLPGALFRCPV VFRQEILVQVIGTLELVGEIEASSMFSLCSSLSISFNSSKHFHLYGSNASLAQVVMKVDVVYEKQMLYLYVLSGIGGLLLLLIFIVLYKVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGKD LFA-1α
(homo sapiens) 37 QECTKFKVSSCRECIESGPGCTWCQKLNFTGPGDPDSIRCDTRPQLLMRGCAADDIMDPTSLAETQEDHNGGQKQLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYSMLDDLRNVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNQFQTEVGKQLISGNLDAPEGGLDA MMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTYEKLTEIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVTHRNQPRGDCDGVQINVPITFQVKVTATECIQEQSFVIRALGFTDIVTVQVLPQCECRCRDQSRDRSLCHGKGFLECGICRCDTGYIGKNCECQTQGRSSQELEGSCRKDNNSIICSGLGDCVCGQCLCHTSDVPGKLIYGQYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQCERTTEGCLNPRRVECSGRGRCRCNVCECHSGYQLPLCQECPGCPSPCGKYISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKGRTCKERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPNIAAIVGGTVAGIVLIGILLLVIWKALIHLSDLREYRRFEKEKLKSQWNNDNPLFKSATTTVMNPKFAES LFA-1β
(homo sapiens) 38 DFLAHHGTDCWTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYNPLFIPVAVMVTAFSGLAFIIWLARRLKKGKKSKRSMNDPY L-selectin
(homo sapiens) 39 FKIETTPESRYLAQIGDSVSLTCSTTGCESPFFSWRTQIDSPLNGKVTNEGTTSTLTMNPVSFGNEHSYLCTATCESRKLEKGIQVEIYSFPKDPEIHLSGPLEAGKPITVKCSVADVYPFDRLEIDLLKGDHLMKSQEFLEDADRKSLETKSLEVTFTPVIEDIGKVLVCRAKLHIDEMDSVPTVRQAVKELQVYISPKNTVISVNPSTKLQEGGSVTMTCSSEGLPAPEIFWSKKLDNGNLQHLSGNATLTLIAMRMEDSGIYVCEGVNLIGKNRKEVELIVQEKPFTVEISPGPRIAAQIGDSVMLTCSV MGCESPSFSWRTQIDSPLSGKVRSEGTNSTLTLSPVSFENEHSYLCTVTCGHKKLEKGIQVELYSFPRDPEIEMSGGLVNGSSVTVSCKVPSVYPLDRLEIELLKGETILENIEFLEDTDMKSLENKSLEMTFIPTIEDTGKALVCQAKLHIDDMEFEPKQRQSTQTLYVNVAPRDTTVLVSPSSILEEGSSVNMTCLSQGFPAPKILWSRQLPNGELQPLSENATLTLISTKMEDSGVYLCEGINQAGRSRKEVELIIQVTPKDIKLTAFPSESVKEGDTVIISCTCGNVPETWIILKKKAETGDTVLKSIDGAYTIRKAQLKDAGVYECESKNKVGSQLRSLTLDVQGRENNKDYFSPELLVLYFASSLIIPAIGMIIYFARKANMKGSYSLVEAQKSKV VCAM-1
(homo sapiens) 40 YNVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIV MGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPETC MVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFTIVIISSSLLLGLIVLLLISYVMWKAGFFKRQYKSILQEENRRDSWSYINSKSND D VLA-4
(homo sapiens) 41 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT IL-2 42 HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS IL-4 43 DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKIL MGTKEH IL-7 44 SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN IL-10 45 GIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS IL-15 46 GITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA IL-17 47 TPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSADVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT CXCL9 48 VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP CXCL10 49 GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS CCL19 fifty SDGGAQDCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQGCRKDRGASKTGKKGKGSKGCKRTERSQTPKGP CCL21 51 QGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHHNTQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL CCL25 52 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Arg Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr Cys Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser αCD16 antibody 3G8 VH 53 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Thr Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys αCD16 antibody 3G8 VL 54 YNLDVRGARSFSPPRAGRHFGYRVLQVGNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPTDGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQECIKGNVDLVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFSDYVKRKDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIIITDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETLHKFASKPASEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGAKDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLASGAPRYQH MGRVLLFQEPQGGGHWSQVQTIHGTQIGSYFGGELCGVDVDQDGETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITALTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQWFGRSIHGVKDLEGDGLADVAVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVHEVECSYSTSNKMKEGVNITICFQIKSLIPQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTPRDQRAQGKDIPPILRPSLHSETWEIPFEKNCGEDKKCEANLRVSFSPARSRALRLTAFASLSVELSLSNLEEDAYWVQLDLHFPPGLSFRKVEMLKPHSQIPVSCEELPEESRLLSRALSCNVSSPIFKAGHSVALQ MMFNTLVNSSWGDSVELHANVTCNNEDSDLLEDNSATTIIPILYPINILIQDQEDSTLYVSFTPKGPKIHQVKHMYQVRIQPSIHDHNIPTLEAVVGVPQPPSEGPITHQWSVQMEPPVPCHYEDLERLPDAAEPCLPGALFRCPV VFRQEILVQVIGTLELVGEIEASSMFSLCSSLSISFNSSKHFHLYGSNASLAQVVMKVDVVYEKQML LFA-1α Extracellular Domain (ECD) 55 QECTKFKVSSCRECIESGPGCTWCQKLNFTGPGDPDSIRCDTRPQLLMRGCAADDIMDPTSLAETQEDHNGGQKQLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYSMLDDLRNVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNQFQTEVGKQLISGNLDAPEGGLDA MMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTYEKLTEIIPKSAVGELSEDSSNVVQLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVTHRNQPRGDCDGVQINVPITFQVKVTATECIQEQSFVIRALGFTDIVTVQVLPQCECRCRDQSRDRSLCHGKGFLECGICRCDTGYIGKNCECQTQGRSSQELEGSCRKDNNSIICSGLGDCVCGQCLCHTSDVPGKLIYGQYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQCERTTEGCLNPRRVECSGRGRCRCNVCECHSGYQLPLCQECPGCPSPCGKYISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKGRTCKERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPN LFA-1β Extracellular Domain (ECD) 56 FKIETTPESRYLAQIGDSVSLTCSTTGCESPFFSWRTQIDSPLNGKVTNEGTTSTLTMNPVSFGNEHSYLCTATCESRKLEKGIQVEIYSFPKDPEIHLSGPLEAGKPITVKCSVADVYPFDRLEIDLLKGDHLMKSQEFLEDADRKSLETKSLEVTFTPVIEDIGKVLVCRAKLHIDEMDSVPTVRQAVKELQVYISPKNTVISVNPSTKLQEGGSVTMTCSSEGLPAPEIFWSKKLDNGNLQHLSGNATLTLIAMRMEDSGIYVCEGVNLIGKNRKEVELIVQEKPFTVEISPGPRIAAQIGDSVMLTCSV VGCESPSFSWRTQIDSPLSGKVRSEGTNSTLTLSPVSFENEHSYLCTVTCGHKKLEKGIQVELYSFPRDPEIEMSGGLVNGSSVTVSCKVPSVYPLDRLEIELLKGETILENIEFLEDTDMKSLENKSLEMTFIPTIEDTGKALVCQAKLHIDDMEFEPKQRQSTQTLYVNVAPRDTTVLVSPSSILEEGSSVNMTCLSQGFPAPKILWSRQLPNGELQPLSENATLTLISTKMEDSGVYLCEGINQAGRSRKEVELIIQVTPKDIKLTAFPSESVKEGDTVIISCTCGNVPETWIILKKKAETGDTVLKSIDGAYTIRKAQLKDAGVYECESKNKVGSQLRSLTLDVQGRENNKDYFSPE VCAM-1 Extracellular Domain (ECD) 57 WTYHYSEKPMNWQRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTWVGTNKSLTEEAENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEGTNLTGIEETTCGPFGNWSSPEPTCQVIQCEPLSAPDLGIMNCSHPLASFSFTSACTFICSEGTELIGKKKTICESSGIWSNPSPICQKLDKSFSMIKEGDYN L-selectin Extracellular Domain (ECD) 58 NVDTESALLYQGPHNTLFGYSVVLHSHGANRWLLVGAPTANWLANASVINPGAIYRCRIGKNPGQTCEQLQLGSPNGEPCGKTCLEERDNQWLGVTLSRQPGENGSIVTCGHRWKNIFYIKNENKLPTGGCYGVPPDLRTELSKRIAPCYQDYVKKFGENFASCQAGISSFYTKDLIV MGAPGSSYWTGSLFVYNITTNKYKAFLDKQNQVKFGSYLGYSVGAGHFRSQHTTEVVGGAPQHEQIGKAYIFSIDEKELNILHEMKGKKLGSYFGASVCAVDLNADGFSDLLVGAPMQSTIREEGRVFVYINSGSGAVMNAMETNLVGSDKYAARFGESIVNLGDIDNDGFEDVAIGAPQEDDLQGAIYIYNGRADGISSTFSQRIEGLQISKSLSMFGQSISGQIDADNNGYVDVAVGAFRSDSAVLLRTRPVVIVDASLSHPESVNRTKFDCVENGWPSVCIDLTLCFSYKGKEVPGYIVLFYNMSLDVNRKAESPPRFYFSSNGTSDVITGSIQVSSREANCRTHQAFMRKDVRDILTPIQIEAAYHLGPHVISKRSTEEFPPLQPILQQKKEKDIMKKTINFARFCAHENCSADLQVSAKIGFLKPHENKTYLAVGSMKTLMLNVSLFNAGDDAYETTLHVKLPVGLYFIKILELEEKQINCEVTDNSGVVQLDCSIGYIYVDHLSRIDISFLLDVSSLSRAEEDLSITVHATCENEEEMDNLKHSRVTVAIPLKYEVKLTVHGFVNPTSFVYGSNDENEPE TCMVEKMNLTFHVINTGNSMAPNVSVEIMVPNSFSPQTDKLFNILDVQTTTGECHFENYQRVCALEQQKSAMQTLKGIVRFLSKTDKRLLYCIKADPHCLNFLCNFGKMESGKEASVHIQLEGRPSILEMDETSALKFEIRATGFPEPNPRVIELNKDENVAHVLLEGLHHQRPKRYFT VLA-4 Extracellular Domain (ECD) 59 MEAGITGTWYNQLGSTFIVTAGADGALTGTYESAVGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS minimal streptavidin
Species: Streptomyces avidinii 60 MEAGITGTWYNQLGSTFIVTAGADGALTGTYVTARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS Mutein Streptavidin Val44-Thr45-Ala46-Arg47
Species: Streptomyces avidinii 61 EAGITGTWYNQLGSTFIVTAGADGALTGTYIGARGNAESRYVLTGRYDSAPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTEANAWKSTLVGHDTFTKVKPSAAS Mutein Streptavidin Ile44-Gly45-Ala-46-Arg47
Species: Streptomyces avidinii

--->

SEQUENCE LIST

<110> JUNO THERAPEUTICS GMBH

SCHMIDT, Thomas

STEMBERGER, Christian

KOWSKI, Tom

PRENTICE, Ken

<120> particulate oligomeric reagents and methods of using the same

<130> 735042008540

<140> not assigned yet

<141> at the same time as

<150> US 62/491,245

<151> 2017-04-27

<160> 61

<170>PatentIn version 3.5

<210> 1

<211> 159

<212> PRT

<213> Streptomyces avidinii

<220>

<221> misc_feature

<223> Streptavidin

<400> 1

Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly

1 5 10 15

Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr

20 25 30

Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser Ala Val Gly

35 40 45

Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro

50 55 60

Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys

65 70 75 80

Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr

85 90 95

Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser

100 105 110

Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp

115 120 125

Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys

130 135 140

Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln

145 150 155

<210> 2

<211> 126

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Minimal streptavidin

<400> 2

Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe

1 5 10 15

Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu Ser

20 25 30

Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 3

<211> 159

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Streptavidin mutein Val44-Thr45-Ala46-Arg47

<400> 3

Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly

1 5 10 15

Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr

20 25 30

Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Val Thr Ala Arg Gly

35 40 45

Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro

50 55 60

Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys

65 70 75 80

Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr

85 90 95

Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser

100 105 110

Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp

115 120 125

Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys

130 135 140

Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln

145 150 155

<210> 4

<211> 126

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Streptavidin mutein Val44-Thr45-Ala46-Arg47

<400> 4

Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe

1 5 10 15

Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Val Thr

20 25 30

Ala Arg Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 5

<211> 159

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Streptavidin mutein Ile44-Gly45-Ala-46-Arg47

<400> 5

Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly

1 5 10 15

Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr

20 25 30

Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Ile Gly Ala Arg Gly

35 40 45

Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro

50 55 60

Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys

65 70 75 80

Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr

85 90 95

Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser

100 105 110

Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val Gly His Asp

115 120 125

Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser Ile Asp Ala Ala Lys

130 135 140

Lys Ala Gly Val Asn Asn Gly Asn Pro Leu Asp Ala Val Gln Gln

145 150 155

<210> 6

<211> 126

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other sign

<223> Streptavidin mutein Ile44-Gly45-Ala-46-Arg47

<400> 6

Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe

1 5 10 15

Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Ile Gly

20 25 30

Ala Arg Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 7

<211> 8

<212> PRT

<213> Faux Sequence

<220>

<223> Streptavidin binding peptide, Strep-tag

<400> 7

Trp Arg His Pro Gln Phe Gly Gly

fifteen

<210> 8

<211> 8

<212> PRT

<213> Faux Sequence

<220>

<223> Strep-tag

<400> 8

Trp Arg His Pro Gln Phe Gly Gly

fifteen

<210> 9

<211> 3

<212> PRT

<213> Faux Sequence

<220>

<223> Streptavidin binding peptide

<220>

<221> OPTION

<222> (3)..(3)

<223> Xaa is selected from glutamine, asparagine and methionine

<400> 9

His ProXaa

one

<210> 10

<211> 4

<212> PRT

<213> Faux Sequence

<220>

<223> Streptavidin binding peptide

<400> 10

His Pro Gln Phe

one

<210> 11

<211> 8

<212> PRT

<213> Faux Sequence

<220>

<223> Streptavidin binding peptide

<220>

<221> OPTION

<222> (1)..(1)

<223> Xaa is Trp, Lys, or Arg;

<220>

<221> OPTION

<222> (2)..(2)

<223> Xaa is any amino acid

<220>

<221> OPTION

<222>(7)..(7)

<223> Xaa is Gly or Glu

<220>

<221> OPTION

<222>(8)..(8)

<223> Xaa is Gly, Lys or Arg

<400> 11

Xaa Xaa His Pro Gln Phe Xaa Xaa

fifteen

<210> 12

<211> 8

<212> PRT

<213> Faux Sequence

<220>

<223> Streptavidin binding peptide

<220>

<221> OPTION

<222> (2)..(2)

<223> Xaa is any amino acid

<220>

<221> OPTION

<222>(7)..(7)

<223> Xaa is Gly or Glu

<220>

<221> OPTION

<222>(8)..(8)

<223> Xaa is Gly, Lys or Arg

<400> 12

Trp Xaa His Pro Gln Phe Xaa Xaa

fifteen

<210> 13

<211> 17

<212> PRT

<213> Faux Sequence

<220>

<223> Serial modules of streptavidin-binding peptide

<220>

<221> OPTION

<222>(9)..(9)

<223> Xaa is any amino acid

<220>

<221> REPEAT

<222>(9)..(9)

<223> Xaa repeated 8 or 12 times

<400> 13

Trp Ser His Pro Gln Phe Glu Lys Xaa Trp Ser His Pro Gln Phe Glu

1 5 10 15

Lys

<210> 14

<211> 20

<212> PRT

<213> Faux Sequence

<220>

<223> Serial modules of streptavidin-binding peptide

<220>

<221> REPEAT

<222> (9)..(12)

<223> repeated 2 or 3 times

<400> 14

Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Trp Ser His Pro

1 5 10 15

Gln Phe Glu Lys

twenty

<210> 15

<211> 30

<212> PRT

<213> Faux Sequence

<220>

<223> Twin-strep-tag

<400> 15

Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Gly Ser Trp Ser His Pro Gln Phe Glu Lys

20 25 30

<210> 16

<211> 30

<212> PRT

<213> Faux Sequence

<220>

<223> Twin-strep-tag

<400> 16

Ser Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

20 25 30

<210> 17

<211> 28

<212> PRT

<213> Faux Sequence

<220>

<223> Twin-strep-tag

<400> 17

Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Trp Ser His Pro Gln Phe Glu Lys

20 25

<210> 18

<211> 24

<212> PRT

<213> Faux Sequence

<220>

<223> Twin-strep-tag

<400> 18

Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Trp Ser His Pro Gln Phe Glu Lys

twenty

<210> 19

<211> 28

<212> PRT

<213> Faux Sequence

<220>

<223> Twin-strep-tag

<400> 19

Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

20 25

<210> 20

<211> 9

<212> PRT

<213> Faux Sequence

<220>

<223> HA-tag

<400> 20

Tyr Pro Tyr Asp Val Pro Asp Tyr Ala

fifteen

<210> 21

<211> 11

<212> PRT

<213> Faux Sequence

<220>

<223> VSV-G-tag

<400> 21

Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Faux Sequence

<220>

<223> HSV tag

<400> 22

Gln Pro Glu Leu Ala Pro Glu Asp Pro Glu Asp

1 5 10

<210> 23

<211> 10

<212> PRT

<213> Faux Sequence

<220>

<223> T7 epitope

<400> 23

Ala Ser Met Thr Gly Gly Glyn Gln Met Gly

1 5 10

<210> 24

<211> 10

<212> PRT

<213> Faux Sequence

<220>

<223> HSV epitope

<400> 24

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 25

<211> 10

<212> PRT

<213> Faux Sequence

<220>

<223> Myc epitope

<400> 25

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 26

<211> 14

<212> PRT

<213> Faux Sequence

<220>

<223> V5-tag

<400> 26

Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr

1 5 10

<210> 27

<211> 126

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Streptavidin mutein Ile44-Gly45-Ala-46-Arg47 and Glu117, Gly120,

Try121 (mutein m1-9)

<400> 27

Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe

1 5 10 15

Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Val Thr

20 25 30

Ala Arg Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Glu Asn Ala Gly Tyr Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 28

<211> 139

<212> PRT

<213> Streptomyces avidinii

<220>

<221> other attribute

<223> Streptavidin mutein Ile44-Gly45-Ala-46-Arg47 and Glu117, Gly120,

Try121 (mutein m1-9)

<400> 28

Asp Pro Ser Lys Asp Ser Lys Ala Gln Val Ser Ala Ala Glu Ala Gly

1 5 10 15

Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe Ile Val Thr

20 25 30

Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Val Thr Ala Arg Gly

35 40 45

Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp Ser Ala Pro

50 55 60

Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val Ala Trp Lys

65 70 75 80

Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser Gly Gln Tyr

85 90 95

Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu Leu Thr Ser

100 105 110

Gly Thr Thr Glu Glu Asn Ala Gly Tyr Ser Thr Leu Val Gly His Asp

115 120 125

Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

130 135

<210> 29

<211> 253

<212> PRT

<213> Faux Sequence

<220>

<223> m13B8.2 Fab heavy chain variable domain

<400> 29

Ala Met Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro

1 5 10 15

Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr

20 25 30

Thr Phe Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Leu Gly Val Ile Trp Ala Ser Gly Ile Thr Asp Tyr Asn Val Pro

50 55 60

Phe Met Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser Gln Val

65 70 75 80

Phe Phe Lys Leu Asn Ser Leu Gln Pro Asp Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Lys Asn Asp Pro Gly Thr Gly Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Gly Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Gly Ser

210 215 220

Ala Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Ser Gly Gly Gly

225 230 235 240

Ser Gly Gly Ser Ala Trp Ser His Pro Gln Phe Glu Lys

245 250

<210> 30

<211> 218

<212> PRT

<213> Faux Sequence

<220>

<223> Light chain variable domain of Fab fragment m13B8.2

<400> 30

Ala Met Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser

1 5 10 15

Val Gly Glu Thr Val Thr Phe Thr Cys Arg Ala Ser Glu Met Ile Tyr

20 25 30

Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu

35 40 45

Leu Val His Asp Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe

50 55 60

Ser Gly Gly Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Thr Leu

65 70 75 80

Gln Pro Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Ala His Tyr Gly Asn

85 90 95

Pro Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Ile

100 105 110

Ala Ala Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys

115 120 125

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

130 135 140

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

145 150 155 160

Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser

165 170 175

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

180 185 190

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

195 200 205

Lys Ser Phe Asn Arg Gly Glu Cys Gly Ser

210 215

<210> 31

<211> 119

<212> PRT

<213> Faux Sequence

<220>

<223> Anti-CD3 OKT3 light chain variable domain

<400> 31

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 32

<211> 106

<212> PRT

<213> Faux Sequence

<220>

<223> Anti-CD3 OKT3 light chain variable domain

<400> 32

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn

100 105

<210> 33

<211> 116

<212> PRT

<213> Faux Sequence

<220>

<223> Heavy chain variable domain of anti-CD28 antibody CD28.3

<400> 33

Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Arg

1 5 10 15

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr Ile Ile His

20 25 30

Trp Ile Lys Leu Arg Ser Gly Gln Gly Leu Glu Trp Ile Gly Trp Phe

35 40 45

Tyr Pro Gly Ser Asn Asp Ile Gln Tyr Asn Ala Lys Phe Lys Gly Lys

50 55 60

Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Val Tyr Met Glu Leu

65 70 75 80

Thr Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Arg

85 90 95

Asp Asp Phe Ser Gly Tyr Asp Ala Leu Pro Tyr Trp Gly Gln Gly Thr

100 105 110

Met Val Thr Val

115

<210> 34

<211> 108

<212> PRT

<213> Faux Sequence

<220>

<223> Anti-CD28 CD28.3 light chain variable domain

<400> 34

Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Val Ser Val Gly

1 5 10 15

Glu Thr Val Thr Ile Thr Cys Arg Thr Asn Glu Asn Ile Tyr Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile

35 40 45

Tyr Ala Ala Thr His Leu Val Glu Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Gly Asn Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Cys

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

<210> 35

<211> 11

<212> PRT

<213> Faux Sequence

<220>

<223> MAT tag

<400> 35

His Asn His Arg His Lys His Gly Gly Gly Cys

1 5 10

<210> 36

<211> 1145

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> LFA-1alpha

<400> 36

Tyr Asn Leu Asp Val Arg Gly Ala Arg Ser Phe Ser Pro Pro Arg Ala

1 5 10 15

Gly Arg His Phe Gly Tyr Arg Val Leu Gln Val Gly Asn Gly Val Ile

20 25 30

Val Gly Ala Pro Gly Glu Gly Asn Ser Thr Gly Ser Leu Tyr Gln Cys

35 40 45

Gln Ser Gly Thr Gly His Cys Leu Pro Val Thr Leu Arg Gly Ser Asn

50 55 60

Tyr Thr Ser Lys Tyr Leu Gly Met Thr Leu Ala Thr Asp Pro Thr Asp

65 70 75 80

Gly Ser Ile Leu Ala Cys Asp Pro Gly Leu Ser Arg Thr Cys Asp Gln

85 90 95

Asn Thr Tyr Leu Ser Gly Leu Cys Tyr Leu Phe Arg Gln Asn Leu Gln

100 105 110

Gly Pro Met Leu Gln Gly Arg Pro Gly Phe Gln Glu Cys Ile Lys Gly

115 120 125

Asn Val Asp Leu Val Phe Leu Phe Asp Gly Ser Met Ser Leu Gln Pro

130 135 140

Asp Glu Phe Gln Lys Ile Leu Asp Phe Met Lys Asp Val Met Lys Lys

145 150 155 160

Leu Ser Asn Thr Ser Tyr Gln Phe Ala Ala Val Gln Phe Ser Thr Ser

165 170 175

Tyr Lys Thr Glu Phe Asp Phe Ser Asp Tyr Val Lys Arg Lys Asp Pro

180 185 190

Asp Ala Leu Leu Lys His Val Lys His Met Leu Leu Leu Thr Asn Thr

195 200 205

Phe Gly Ala Ile Asn Tyr Val Ala Thr Glu Val Phe Arg Glu Glu Leu

210 215 220

Gly Ala Arg Pro Asp Ala Thr Lys Val Leu Ile Ile Ile Thr Asp Gly

225 230 235 240

Glu Ala Thr Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Ile Ile Arg

245 250 255

Tyr Ile Ile Gly Ile Gly Lys His Phe Gln Thr Lys Glu Ser Gln Glu

260 265 270

Thr Leu His Lys Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Lys Ile

275 280 285

Leu Asp Thr Phe Glu Lys Leu Lys Asp Leu Phe Thr Glu Leu Gln Lys

290 295 300

Lys Ile Tyr Val Ile Glu Gly Thr Ser Lys Gln Asp Leu Thr Ser Phe

305 310 315 320

Asn Met Glu Leu Ser Ser Ser Gly Ile Ser Ala Asp Leu Ser Arg Gly

325 330 335

His Ala Val Val Gly Ala Val Gly Ala Lys Asp Trp Ala Gly Gly Phe

340 345 350

Leu Asp Leu Lys Ala Asp Leu Gln Asp Asp Thr Phe Ile Gly Asn Glu

355 360 365

Pro Leu Thr Pro Glu Val Arg Ala Gly Tyr Leu Gly Tyr Thr Val Thr

370 375 380

Trp Leu Pro Ser Arg Gln Lys Thr Ser Leu Leu Ala Ser Gly Ala Pro

385 390 395 400

Arg Tyr Gln His Met Gly Arg Val Leu Leu Phe Gln Glu Pro Gln Gly

405 410 415

Gly Gly His Trp Ser Gln Val Gln Thr Ile His Gly Thr Gln Ile Gly

420 425 430

Ser Tyr Phe Gly Gly Glu Leu Cys Gly Val Asp Val Asp Gln Asp Gly

435 440 445

Glu Thr Glu Leu Leu Leu Ile Gly Ala Pro Leu Phe Tyr Gly Glu Gln

450 455 460

Arg Gly Gly Arg Val Phe Ile Tyr Gln Arg Arg Gln Leu Gly Phe Glu

465 470 475 480

Glu Val Ser Glu Leu Gln Gly Asp Pro Gly Tyr Pro Leu Gly Arg Phe

485 490 495

Gly Glu Ala Ile Thr Ala Leu Thr Asp Ile Asn Gly Asp Gly Leu Val

500 505 510

Asp Val Ala Val Gly Ala Pro Leu Glu Glu Gln Gly Ala Val Tyr Ile

515 520 525

Phe Asn Gly Arg His Gly Gly Leu Ser Pro Gln Pro Ser Gln Arg Ile

530 535 540

Glu Gly Thr Gln Val Leu Ser Gly Ile Gln Trp Phe Gly Arg Ser Ile

545 550 555 560

His Gly Val Lys Asp Leu Glu Gly Asp Gly Leu Ala Asp Val Ala Val

565 570 575

Gly Ala Glu Ser Gln Met Ile Val Leu Ser Ser Arg Pro Val Val Asp

580 585 590

Met Val Thr Leu Met Ser Phe Ser Pro Ala Glu Ile Pro Val His Glu

595 600 605

Val Glu Cys Ser Tyr Ser Thr Ser Asn Lys Met Lys Glu Gly Val Asn

610 615 620

Ile Thr Ile Cys Phe Gln Ile Lys Ser Leu Ile Pro Gln Phe Gln Gly

625 630 635 640

Arg Leu Val Ala Asn Leu Thr Tyr Thr Leu Gln Leu Asp Gly His Arg

645 650 655

Thr Arg Arg Arg Gly Leu Phe Pro Gly Gly Arg His Glu Leu Arg Arg

660 665 670

Asn Ile Ala Val Thr Thr Ser Met Ser Cys Thr Asp Phe Ser Phe His

675 680 685

Phe Pro Val Cys Val Gln Asp Leu Ile Ser Pro Ile Asn Val Ser Leu

690 695 700

Asn Phe Ser Leu Trp Glu Glu Glu Gly Thr Pro Arg Asp Gln Arg Ala

705 710 715 720

Gln Gly Lys Asp Ile Pro Pro Ile Leu Arg Pro Ser Leu His Ser Glu

725 730 735

Thr Trp Glu Ile Pro Phe Glu Lys Asn Cys Gly Glu Asp Lys Lys Cys

740 745 750

Glu Ala Asn Leu Arg Val Ser Phe Ser Pro Ala Arg Ser Arg Ala Leu

755 760 765

Arg Leu Thr Ala Phe Ala Ser Leu Ser Val Glu Leu Ser Leu Ser Asn

770 775 780

Leu Glu Glu Asp Ala Tyr Trp Val Gln Leu Asp Leu His Phe Pro Pro

785 790 795 800

Gly Leu Ser Phe Arg Lys Val Glu Met Leu Lys Pro His Ser Gln Ile

805 810 815

Pro Val Ser Cys Glu Glu Leu Pro Glu Glu Ser Arg Leu Leu Ser Arg

820 825 830

Ala Leu Ser Cys Asn Val Ser Ser Pro Ile Phe Lys Ala Gly His Ser

835 840 845

Val Ala Leu Gln Met Met Phe Asn Thr Leu Val Asn Ser Ser Trp Gly

850 855 860

Asp Ser Val Glu Leu His Ala Asn Val Thr Cys Asn Asn Glu Asp Ser

865 870 875 880

Asp Leu Leu Glu Asp Asn Ser Ala Thr Thr Ile Ile Pro Ile Leu Tyr

885 890 895

Pro Ile Asn Ile Leu Ile Gln Asp Gln Glu Asp Ser Thr Leu Tyr Val

900 905 910

Ser Phe Thr Pro Lys Gly Pro Lys Ile His Gln Val Lys His Met Tyr

915 920 925

Gln Val Arg Ile Gln Pro Ser Ile His Asp His Asn Ile Pro Thr Leu

930 935 940

Glu Ala Val Val Gly Val Pro Gln Pro Pro Ser Glu Gly Pro Ile Thr

945 950 955 960

His Gln Trp Ser Val Gln Met Glu Pro Pro Val Pro Cys His Tyr Glu

965 970 975

Asp Leu Glu Arg Leu Pro Asp Ala Ala Glu Pro Cys Leu Pro Gly Ala

980 985 990

Leu Phe Arg Cys Pro Val Val Phe Arg Gln Glu Ile Leu Val Gln Val

995 1000 1005

Ile Gly Thr Leu Glu Leu Val Gly Glu Ile Glu Ala Ser Ser Met

1010 1015 1020

Phe Ser Leu Cys Ser Ser Leu Ser Ile Ser Phe Asn Ser Ser Lys

1025 1030 1035

His Phe His Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gln Val Val

1040 1045 1050

Met Lys Val Asp Val Val Tyr Glu Lys Gln Met Leu Tyr Leu Tyr

1055 1060 1065

Val Leu Ser Gly Ile Gly Gly Leu Leu Leu Leu Leu Leu Ile Phe

1070 1075 1080

Ile Val Leu Tyr Lys Val Gly Phe Phe Lys Arg Asn Leu Lys Glu

1085 1090 1095

Lys Met Glu Ala Gly Arg Gly Val Pro Asn Gly Ile Pro Ala Glu

1100 1105 1110

Asp Ser Glu Gln Leu Ala Ser Gly Gln Glu Ala Gly Asp Pro Gly

1115 1120 1125

Cys Leu Lys Pro Leu His Glu Lys Asp Ser Glu Ser Gly Gly Gly

1130 1135 1140

Lys Asp

1145

<210> 37

<211> 747

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> LFA-1beta

<400> 37

Gln Glu Cys Thr Lys Phe Lys Val Ser Ser Cys Arg Glu Cys Ile Glu

1 5 10 15

Ser Gly Pro Gly Cys Thr Trp Cys Gln Lys Leu Asn Phe Thr Gly Pro

20 25 30

Gly Asp Pro Asp Ser Ile Arg Cys Asp Thr Arg Pro Gln Leu Leu Met

35 40 45

Arg Gly Cys Ala Ala Asp Asp Ile Met Asp Pro Thr Ser Leu Ala Glu

50 55 60

Thr Gln Glu Asp His Asn Gly Gly Gln Lys Gln Leu Ser Pro Gln Lys

65 70 75 80

Val Thr Leu Tyr Leu Arg Pro Gly Gln Ala Ala Ala Phe Asn Val Thr

85 90 95

Phe Arg Arg Ala Lys Gly Tyr Pro Ile Asp Leu Tyr Tyr Leu Met Asp

100 105 110

Leu Ser Tyr Ser Met Leu Asp Asp Leu Arg Asn Val Lys Lys Leu Gly

115 120 125

Gly Asp Leu Leu Arg Ala Leu Asn Glu Ile Thr Glu Ser Gly Arg Ile

130 135 140

Gly Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val Asn Thr

145 150 155 160

His Pro Asp Lys Leu Arg Asn Pro Cys Pro Asn Lys Glu Lys Glu Cys

165 170 175

Gln Pro Pro Phe Ala Phe Arg His Val Leu Lys Leu Thr Asn Asn Ser

180 185 190

Asn Gln Phe Gln Thr Glu Val Gly Lys Gln Leu Ile Ser Gly Asn Leu

195 200 205

Asp Ala Pro Glu Gly Gly Leu Asp Ala Met Met Gln Val Ala Ala Cys

210 215 220

Pro Glu Glu Ile Gly Trp Arg Asn Val Thr Arg Leu Leu Val Phe Ala

225 230 235 240

Thr Asp Asp Gly Phe His Phe Ala Gly Asp Gly Lys Leu Gly Ala Ile

245 250 255

Leu Thr Pro Asn Asp Gly Arg Cys His Leu Glu Asp Asn Leu Tyr Lys

260 265 270

Arg Ser Asn Glu Phe Asp Tyr Pro Ser Val Gly Gln Leu Ala His Lys

275 280 285

Leu Ala Glu Asn Asn Ile Gln Pro Ile Phe Ala Val Thr Ser Arg Met

290 295 300

Val Lys Thr Tyr Glu Lys Leu Thr Glu Ile Ile Pro Lys Ser Ala Val

305 310 315 320

Gly Glu Leu Ser Glu Asp Ser Ser Asn Val Val Gln Leu Ile Lys Asn

325 330 335

Ala Tyr Asn Lys Leu Ser Ser Arg Val Phe Leu Asp His Asn Ala Leu

340 345 350

Pro Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Val

355 360 365

Thr His Arg Asn Gln Pro Arg Gly Asp Cys Asp Gly Val Gln Ile Asn

370 375 380

Val Pro Ile Thr Phe Gln Val Lys Val Thr Ala Thr Glu Cys Ile Gln

385 390 395 400

Glu Gln Ser Phe Val Ile Arg Ala Leu Gly Phe Thr Asp Ile Val Thr

405 410 415

Val Gln Val Leu Pro Gln Cys Glu Cys Arg Cys Arg Asp Gln Ser Arg

420 425 430

Asp Arg Ser Leu Cys His Gly Lys Gly Phe Leu Glu Cys Gly Ile Cys

435 440 445

Arg Cys Asp Thr Gly Tyr Ile Gly Lys Asn Cys Glu Cys Gln Thr Gln

450 455 460

Gly Arg Ser Ser Gln Glu Leu Glu Gly Ser Cys Arg Lys Asp Asn Asn

465 470 475 480

Ser Ile Ile Cys Ser Gly Leu Gly Asp Cys Val Cys Gly Gln Cys Leu

485 490 495

Cys His Thr Ser Asp Val Pro Gly Lys Leu Ile Tyr Gly Gln Tyr Cys

500 505 510

Glu Cys Asp Thr Ile Asn Cys Glu Arg Tyr Asn Gly Gln Val Cys Gly

515 520 525

Gly Pro Gly Arg Gly Leu Cys Phe Cys Gly Lys Cys Arg Cys His Pro

530 535 540

Gly Phe Glu Gly Ser Ala Cys Gln Cys Glu Arg Thr Thr Glu Gly Cys

545 550 555 560

Leu Asn Pro Arg Arg Val Glu Cys Ser Gly Arg Gly Arg Cys Arg Cys

565 570 575

Asn Val Cys Glu Cys His Ser Gly Tyr Gln Leu Pro Leu Cys Gln Glu

580 585 590

Cys Pro Gly Cys Pro Ser Pro Cys Gly Lys Tyr Ile Ser Cys Ala Glu

595 600 605

Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly Lys Asn Cys Ser Ala Ala

610 615 620

Cys Pro Gly Leu Gln Leu Ser Asn Asn Pro Val Lys Gly Arg Thr Cys

625 630 635 640

Lys Glu Arg Asp Ser Glu Gly Cys Trp Val Ala Tyr Thr Leu Glu Gln

645 650 655

Gln Asp Gly Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg Glu

660 665 670

Cys Val Ala Gly Pro Asn Ile Ala Ala Ile Val Gly Gly Thr Val Ala

675 680 685

Gly Ile Val Leu Ile Gly Ile Leu Leu Leu Val Ile Trp Lys Ala Leu

690 695 700

Ile His Leu Ser Asp Leu Arg Glu Tyr Arg Arg Phe Glu Lys Glu Lys

705 710 715 720

Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro Leu Phe Lys Ser Ala Thr

725 730 735

Thr Thr Val Met Asn Pro Lys Phe Ala Glu Ser

740 745

<210> 38

<211> 344

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> L-selectin

<400> 38

Asp Phe Leu Ala His His Gly Thr Asp Cys Trp Thr Tyr His Tyr Ser

1 5 10 15

Glu Lys Pro Met Asn Trp Gln Arg Ala Arg Arg Phe Cys Arg Asp Asn

20 25 30

Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys Ala Glu Ile Glu Tyr Leu

35 40 45

Glu Lys Thr Leu Pro Phe Ser Arg Ser Tyr Tyr Trp Ile Gly Ile Arg

50 55 60

Lys Ile Gly Gly Ile Trp Thr Trp Val Gly Thr Asn Lys Ser Leu Thr

65 70 75 80

Glu Glu Ala Glu Asn Trp Gly Asp Gly Glu Pro Asn Asn Lys Lys Asn

85 90 95

Lys Glu Asp Cys Val Glu Ile Tyr Ile Lys Arg Asn Lys Asp Ala Gly

100 105 110

Lys Trp Asn Asp Asp Ala Cys His Lys Leu Lys Ala Ala Leu Cys Tyr

115 120 125

Thr Ala Ser Cys Gln Pro Trp Ser Cys Ser Gly His Gly Glu Cys Val

130 135 140

Glu Ile Ile Asn Asn Tyr Thr Cys Asn Cys Asp Val Gly Tyr Tyr Gly

145 150 155 160

Pro Gln Cys Gln Phe Val Ile Gln Cys Glu Pro Leu Glu Ala Pro Glu

165 170 175

Leu Gly Thr Met Asp Cys Thr His Pro Leu Gly Asn Phe Ser Phe Ser

180 185 190

Ser Gln Cys Ala Phe Ser Cys Ser Glu Gly Thr Asn Leu Thr Gly Ile

195 200 205

Glu Glu Thr Thr Cys Gly Pro Phe Gly Asn Trp Ser Ser Pro Glu Pro

210 215 220

Thr Cys Gln Val Ile Gln Cys Glu Pro Leu Ser Ala Pro Asp Leu Gly

225 230 235 240

Ile Met Asn Cys Ser His Pro Leu Ala Ser Phe Ser Phe Thr Ser Ala

245 250 255

Cys Thr Phe Ile Cys Ser Glu Gly Thr Glu Leu Ile Gly Lys Lys Lys

260 265 270

Thr Ile Cys Glu Ser Ser Gly Ile Trp Ser Asn Pro Ser Pro Ile Cys

275 280 285

Gln Lys Leu Asp Lys Ser Phe Ser Met Ile Lys Glu Gly Asp Tyr Asn

290 295 300

Pro Leu Phe Ile Pro Val Ala Val Met Val Thr Ala Phe Ser Gly Leu

305 310 315 320

Ala Phe Ile Ile Trp Leu Ala Arg Arg Leu Lys Lys Gly Lys Lys Ser

325 330 335

Lys Arg Ser Met Asn Asp Pro Tyr

340

<210> 39

<211> 715

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> VCAM-1

<400> 39

Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Ala Gln Ile Gly

1 5 10 15

Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe

20 25 30

Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Asn Gly Lys Val Thr

35 40 45

Asn Glu Gly Thr Thr Ser Thr Leu Thr Met Asn Pro Val Ser Phe Gly

50 55 60

Asn Glu His Ser Tyr Leu Cys Thr Ala Thr Cys Glu Ser Arg Lys Leu

65 70 75 80

Glu Lys Gly Ile Gln Val Glu Ile Tyr Ser Phe Pro Lys Asp Pro Glu

85 90 95

Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys

100 105 110

Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu

115 120 125

Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala

130 135 140

Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro

145 150 155 160

Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Leu His

165 170 175

Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu

180 185 190

Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro

195 200 205

Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met Thr Cys Ser Ser

210 215 220

Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys Lys Leu Asp Asn

225 230 235 240

Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala

245 250 255

Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu

260 265 270

Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val Gln Glu Lys Pro

275 280 285

Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala Ala Gln Ile Gly

290 295 300

Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser

305 310 315 320

Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg

325 330 335

Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu

340 345 350

Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu

355 360 365

Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Arg Asp Pro Glu

370 375 380

Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser

385 390 395 400

Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu

405 410 415

Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr

420 425 430

Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro

435 440 445

Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His

450 455 460

Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr

465 470 475 480

Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro

485 490 495

Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser

500 505 510

Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn

515 520 525

Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser

530 535 540

Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln

545 550 555 560

Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro

565 570 575

Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly

580 585 590

Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp

595 600 605

Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser

610 615 620

Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly

625 630 635 640

Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser

645 650 655

Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser

660 665 670

Pro Glu Leu Leu Val Leu Tyr Phe Ala Ser Ser Leu Ile Ile Pro Ala

675 680 685

Ile Gly Met Ile Ile Tyr Phe Ala Arg Lys Ala Asn Met Lys Gly Ser

690 695 700

Tyr Ser Leu Val Glu Ala Gln Lys Ser Lys Val

705 710 715

<210> 40

<211> 999

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> VLA-4

<400> 40

Tyr Asn Val Asp Thr Glu Ser Ala Leu Leu Tyr Gln Gly Pro His Asn

1 5 10 15

Thr Leu Phe Gly Tyr Ser Val Val Leu His Ser His Gly Ala Asn Arg

20 25 30

Trp Leu Leu Val Gly Ala Pro Thr Ala Asn Trp Leu Ala Asn Ala Ser

35 40 45

Val Ile Asn Pro Gly Ala Ile Tyr Arg Cys Arg Ile Gly Lys Asn Pro

50 55 60

Gly Gln Thr Cys Glu Gln Leu Gln Leu Gly Ser Pro Asn Gly Glu Pro

65 70 75 80

Cys Gly Lys Thr Cys Leu Glu Glu Arg Asp Asn Gln Trp Leu Gly Val

85 90 95

Thr Leu Ser Arg Gln Pro Gly Glu Asn Gly Ser Ile Val Thr Cys Gly

100 105 110

His Arg Trp Lys Asn Ile Phe Tyr Ile Lys Asn Glu Asn Lys Leu Pro

115 120 125

Thr Gly Gly Cys Tyr Gly Val Pro Pro Asp Leu Arg Thr Glu Leu Ser

130 135 140

Lys Arg Ile Ala Pro Cys Tyr Gln Asp Tyr Val Lys Lys Phe Gly Glu

145 150 155 160

Asn Phe Ala Ser Cys Gln Ala Gly Ile Ser Ser Phe Tyr Thr Lys Asp

165 170 175

Leu Ile Val Met Gly Ala Pro Gly Ser Ser Tyr Trp Thr Gly Ser Leu

180 185 190

Phe Val Tyr Asn Ile Thr Thr Asn Lys Tyr Lys Ala Phe Leu Asp Lys

195 200 205

Gln Asn Gln Val Lys Phe Gly Ser Tyr Leu Gly Tyr Ser Val Gly Ala

210 215 220

Gly His Phe Arg Ser Gln His Thr Thr Glu Val Val Gly Gly Ala Pro

225 230 235 240

Gln His Glu Gln Ile Gly Lys Ala Tyr Ile Phe Ser Ile Asp Glu Lys

245 250 255

Glu Leu Asn Ile Leu His Glu Met Lys Gly Lys Lys Leu Gly Ser Tyr

260 265 270

Phe Gly Ala Ser Val Cys Ala Val Asp Leu Asn Ala Asp Gly Phe Ser

275 280 285

Asp Leu Leu Val Gly Ala Pro Met Gln Ser Thr Ile Arg Glu Glu Gly

290 295 300

Arg Val Phe Val Tyr Ile Asn Ser Gly Ser Gly Ala Val Met Asn Ala

305 310 315 320

Met Glu Thr Asn Leu Val Gly Ser Asp Lys Tyr Ala Ala Arg Phe Gly

325 330 335

Glu Ser Ile Val Asn Leu Gly Asp Ile Asp Asn Asp Gly Phe Glu Asp

340 345 350

Val Ala Ile Gly Ala Pro Gln Glu Asp Asp Leu Gln Gly Ala Ile Tyr

355 360 365

Ile Tyr Asn Gly Arg Ala Asp Gly Ile Ser Ser Thr Phe Ser Gln Arg

370 375 380

Ile Glu Gly Leu Gln Ile Ser Lys Ser Leu Ser Met Phe Gly Gln Ser

385 390 395 400

Ile Ser Gly Gln Ile Asp Ala Asp Asn Asn Gly Tyr Val Asp Val Ala

405 410 415

Val Gly Ala Phe Arg Ser Asp Ser Ala Val Leu Leu Arg Thr Arg Pro

420 425 430

Val Val Ile Val Asp Ala Ser Leu Ser His Pro Glu Ser Val Asn Arg

435 440 445

Thr Lys Phe Asp Cys Val Glu Asn Gly Trp Pro Ser Val Cys Ile Asp

450 455 460

Leu Thr Leu Cys Phe Ser Tyr Lys Gly Lys Glu Val Pro Gly Tyr Ile

465 470 475 480

Val Leu Phe Tyr Asn Met Ser Leu Asp Val Asn Arg Lys Ala Glu Ser

485 490 495

Pro Pro Arg Phe Tyr Phe Ser Ser Asn Gly Thr Ser Asp Val Ile Thr

500 505 510

Gly Ser Ile Gln Val Ser Ser Arg Glu Ala Asn Cys Arg Thr His Gln

515 520 525

Ala Phe Met Arg Lys Asp Val Arg Asp Ile Leu Thr Pro Ile Gln Ile

530 535 540

Glu Ala Ala Tyr His Leu Gly Pro His Val Ile Ser Lys Arg Ser Thr

545 550 555 560

Glu Glu Phe Pro Pro Leu Gln Pro Ile Leu Gln Gln Lys Lys Glu Lys

565 570 575

Asp Ile Met Lys Lys Thr Ile Asn Phe Ala Arg Phe Cys Ala His Glu

580 585 590

Asn Cys Ser Ala Asp Leu Gln Val Ser Ala Lys Ile Gly Phe Leu Lys

595 600 605

Pro His Glu Asn Lys Thr Tyr Leu Ala Val Gly Ser Met Lys Thr Leu

610 615 620

Met Leu Asn Val Ser Leu Phe Asn Ala Gly Asp Asp Ala Tyr Glu Thr

625 630 635 640

Thr Leu His Val Lys Leu Pro Val Gly Leu Tyr Phe Ile Lys Ile Leu

645 650 655

Glu Leu Glu Glu Lys Gln Ile Asn Cys Glu Val Thr Asp Asn Ser Gly

660 665 670

Val Val Gln Leu Asp Cys Ser Ile Gly Tyr Ile Tyr Val Asp His Leu

675 680 685

Ser Arg Ile Asp Ile Ser Phe Leu Leu Asp Val Ser Ser Leu Ser Arg

690 695 700

Ala Glu Glu Asp Leu Ser Ile Thr Val His Ala Thr Cys Glu Asn Glu

705 710 715 720

Glu Glu Met Asp Asn Leu Lys His Ser Arg Val Thr Val Ala Ile Pro

725 730 735

Leu Lys Tyr Glu Val Lys Leu Thr Val His Gly Phe Val Asn Pro Thr

740 745 750

Ser Phe Val Tyr Gly Ser Asn Asp Glu Asn Glu Pro Glu Thr Cys Met

755 760 765

Val Glu Lys Met Asn Leu Thr Phe His Val Ile Asn Thr Gly Asn Ser

770 775 780

Met Ala Pro Asn Val Ser Val Glu Ile Met Val Pro Asn Ser Phe Ser

785 790 795 800

Pro Gln Thr Asp Lys Leu Phe Asn Ile Leu Asp Val Gln Thr Thr Thr

805 810 815

Gly Glu Cys His Phe Glu Asn Tyr Gln Arg Val Cys Ala Leu Glu Gln

820 825 830

Gln Lys Ser Ala Met Gln Thr Leu Lys Gly Ile Val Arg Phe Leu Ser

835 840 845

Lys Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys Ala Asp Pro His Cys

850 855 860

Leu Asn Phe Leu Cys Asn Phe Gly Lys Met Glu Ser Gly Lys Glu Ala

865 870 875 880

Ser Val His Ile Gln Leu Glu Gly Arg Pro Ser Ile Leu Glu Met Asp

885 890 895

Glu Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala Thr Gly Phe Pro Glu

900 905 910

Pro Asn Pro Arg Val Ile Glu Leu Asn Lys Asp Glu Asn Val Ala His

915 920 925

Val Leu Leu Glu Gly Leu His His Gln Arg Pro Lys Arg Tyr Phe Thr

930 935 940

Ile Val Ile Ile Ser Ser Ser Leu Leu Leu Gly Leu Ile Val Leu Leu

945 950 955 960

Leu Ile Ser Tyr Val Met Trp Lys Ala Gly Phe Phe Lys Arg Gln Tyr

965 970 975

Lys Ser Ile Leu Gln Glu Glu Asn Arg Arg Asp Ser Trp Ser Tyr Ile

980 985 990

Asn Ser Lys Ser Asn Asp Asp

995

<210> 41

<211> 133

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-2

<400> 41

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 42

<211> 129

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-4

<400> 42

His Lys Cys Asp Ile Thr Leu Gln Glu Ile Ile Lys Thr Leu Asn Ser

1 5 10 15

Leu Thr Glu Gln Lys Thr Leu Cys Thr Glu Leu Thr Val Thr Asp Ile

20 25 30

Phe Ala Ala Ser Lys Asn Thr Thr Glu Lys Glu Thr Phe Cys Arg Ala

35 40 45

Ala Thr Val Leu Arg Gln Phe Tyr Ser His His Glu Lys Asp Thr Arg

50 55 60

Cys Leu Gly Ala Thr Ala Gln Gln Phe His Arg His Lys Gln Leu Ile

65 70 75 80

Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu

85 90 95

Asn Ser Cys Pro Val Lys Glu Ala Asn Gln Ser Thr Leu Glu Asn Phe

100 105 110

Leu Glu Arg Leu Lys Thr Ile Met Arg Glu Lys Tyr Ser Lys Cys Ser

115 120 125

Ser

<210> 43

<211> 152

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-7

<400> 43

Asp Cys Asp Ile Glu Gly Lys Asp Gly Lys Gln Tyr Glu Ser Val Leu

1 5 10 15

Met Val Ser Ile Asp Gln Leu Leu Asp Ser Met Lys Glu Ile Gly Ser

20 25 30

Asn Cys Leu Asn Asn Glu Phe Asn Phe Phe Lys Arg His Ile Cys Asp

35 40 45

Ala Asn Lys Glu Gly Met Phe Leu Phe Arg Ala Ala Arg Lys Leu Arg

50 55 60

Gln Phe Leu Lys Met Asn Ser Thr Gly Asp Phe Asp Leu His Leu Leu

65 70 75 80

Lys Val Ser Glu Gly Thr Thr Ile Leu Leu Asn Cys Thr Gly Gln Val

85 90 95

Lys Gly Arg Lys Pro Ala Ala Leu Gly Glu Ala Gln Pro Thr Lys Ser

100 105 110

Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys Leu Asn Asp Leu

115 120 125

Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr Cys Trp Asn Lys

130 135 140

Ile Leu Met Gly Thr Lys Glu His

145 150

<210> 44

<211> 160

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-10

<400> 44

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

<210> 45

<211> 133

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-15

<400> 45

Gly Ile His Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys

1 5 10 15

Thr Glu Ala Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu

20 25 30

Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser

35 40 45

Asp Val His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu

50 55 60

Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp

65 70 75 80

Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn

85 90 95

Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu

100 105 110

Lys Asn Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met

115 120 125

Phe Ile Asn Thr Ser

130

<210> 46

<211> 132

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> IL-17

<400> 46

Gly Ile Thr Ile Pro Arg Asn Pro Gly Cys Pro Asn Ser Glu Asp Lys

1 5 10 15

Asn Phe Pro Arg Thr Val Met Val Asn Leu Asn Ile His Asn Arg Asn

20 25 30

Thr Asn Thr Asn Pro Lys Arg Ser Ser Asp Tyr Tyr Asn Arg Ser Thr

35 40 45

Ser Pro Trp Asn Leu His Arg Asn Glu Asp Pro Glu Arg Tyr Pro Ser

50 55 60

Val Ile Trp Glu Ala Lys Cys Arg His Leu Gly Cys Ile Asn Ala Asp

65 70 75 80

Gly Asn Val Asp Tyr His Met Asn Ser Val Pro Ile Gln Gln Glu Ile

85 90 95

Leu Val Leu Arg Arg Glu Pro Pro His Cys Pro Asn Ser Phe Arg Leu

100 105 110

Glu Lys Ile Leu Val Ser Val Gly Cys Thr Cys Val Thr Pro Ile Val

115 120 125

His His Val Ala

130

<210> 47

<211> 103

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> CXCL9

<400> 47

Thr Pro Val Val Arg Lys Gly Arg Cys Ser Cys Ile Ser Thr Asn Gln

1 5 10 15

Gly Thr Ile His Leu Gln Ser Leu Lys Asp Leu Lys Gln Phe Ala Pro

20 25 30

Ser Pro Ser Cys Glu Lys Ile Glu Ile Ile Ala Thr Leu Lys Asn Gly

35 40 45

Val Gln Thr Cys Leu Asn Pro Asp Ser Ala Asp Val Lys Glu Leu Ile

50 55 60

Lys Lys Trp Glu Lys Gln Val Ser Gln Lys Lys Lys Gln Lys Asn Gly

65 70 75 80

Lys Lys His Gln Lys Lys Lys Val Leu Lys Val Arg Lys Ser Gln Arg

85 90 95

Ser Arg Gln Lys Lys Thr Thr

100

<210> 48

<211> 77

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> CXCL10

<400> 48

Val Pro Leu Ser Arg Thr Val Arg Cys Thr Cys Ile Ser Ile Ser Asn

1 5 10 15

Gln Pro Val Asn Pro Arg Ser Leu Glu Lys Leu Glu Ile Ile Pro Ala

20 25 30

Ser Gln Phe Cys Pro Arg Val Glu Ile Ile Ala Thr Met Lys Lys Lys

35 40 45

Gly Glu Lys Arg Cys Leu Asn Pro Glu Ser Lys Ala Ile Lys Asn Leu

50 55 60

Leu Lys Ala Val Ser Lys Glu Arg Ser Lys Arg Ser Pro

65 70 75

<210> 49

<211> 77

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> CCL19

<400> 49

Gly Thr Asn Asp Ala Glu Asp Cys Cys Leu Ser Val Thr Gln Lys Pro

1 5 10 15

Ile Pro Gly Tyr Ile Val Arg Asn Phe His Tyr Leu Leu Ile Lys Asp

20 25 30

Gly Cys Arg Val Pro Ala Val Val Phe Thr Thr Leu Arg Gly Arg Gln

35 40 45

Leu Cys Ala Pro Pro Asp Gln Pro Trp Val Glu Arg Ile Ile Gln Arg

50 55 60

Leu Gln Arg Thr Ser Ala Lys Met Lys Arg Arg Ser Ser

65 70 75

<210> 50

<211> 111

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> CCL21

<400> 50

Ser Asp Gly Gly Ala Gln Asp Cys Cys Leu Lys Tyr Ser Gln Arg Lys

1 5 10 15

Ile Pro Ala Lys Val Val Arg Ser Tyr Arg Lys Gln Glu Pro Ser Leu

20 25 30

Gly Cys Ser Ile Pro Ala Ile Leu Phe Leu Pro Arg Lys Arg Ser Gln

35 40 45

Ala Glu Leu Cys Ala Asp Pro Lys Glu Leu Trp Val Gln Gln Leu Met

50 55 60

Gln His Leu Asp Lys Thr Pro Ser Pro Gln Lys Pro Ala Gln Gly Cys

65 70 75 80

Arg Lys Asp Arg Gly Ala Ser Lys Thr Gly Lys Lys Gly Lys Gly Ser

85 90 95

Lys Gly Cys Lys Arg Thr Glu Arg Ser Gln Thr Pro Lys Gly Pro

100 105 110

<210> 51

<211> 127

<212> PRT

<213> Homo sapiens

<220>

<221> OTHER SIGN

<223> CCL25

<400> 51

Gln Gly Val Phe Glu Asp Cys Cys Leu Ala Tyr His Tyr Pro Ile Gly

1 5 10 15

Trp Ala Val Leu Arg Arg Ala Trp Thr Tyr Arg Ile Gln Glu Val Ser

20 25 30

Gly Ser Cys Asn Leu Pro Ala Ala Ile Phe Tyr Leu Pro Lys Arg His

35 40 45

Arg Lys Val Cys Gly Asn Pro Lys Ser Arg Glu Val Gln Arg Ala Met

50 55 60

Lys Leu Leu Asp Ala Arg Asn Lys Val Phe Ala Lys Leu His His Asn

65 70 75 80

Thr Gln Thr Phe Gln Ala Gly Pro His Ala Val Lys Lys Leu Ser Ser

85 90 95

Gly Asn Ser Lys Leu Ser Ser Ser Lys Phe Ser Asn Pro Ile Ser Ser

100 105 110

Ser Lys Arg Asn Val Ser Leu Leu Ile Ser Ala Asn Ser Gly Leu

115 120 125

<210> 52

<211> 118

<212> PRT

<213> Faux Sequence

<220>

<223> 3G8 VH anti-CD16 antibody

<400> 52

Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Arg Thr Ser

20 25 30

Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu

35 40 45

Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala

50 55 60

Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val

65 70 75 80

Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr

85 90 95

Cys Ala Gln Ile Asn Pro Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 53

<211> 111

<212> PRT

<213> Faux Sequence

<220>

<223> anti-CD16 antibody 3G8 VL

<400> 53

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Phe Asp

20 25 30

Gly Asp Ser Phe Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Thr Thr Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 54

<211> 1065

<212> PRT

<213> Faux Sequence

<220>

<223> Extracellular domain (ECD) LFA-1alpha

<400> 54

Tyr Asn Leu Asp Val Arg Gly Ala Arg Ser Phe Ser Pro Pro Arg Ala

1 5 10 15

Gly Arg His Phe Gly Tyr Arg Val Leu Gln Val Gly Asn Gly Val Ile

20 25 30

Val Gly Ala Pro Gly Glu Gly Asn Ser Thr Gly Ser Leu Tyr Gln Cys

35 40 45

Gln Ser Gly Thr Gly His Cys Leu Pro Val Thr Leu Arg Gly Ser Asn

50 55 60

Tyr Thr Ser Lys Tyr Leu Gly Met Thr Leu Ala Thr Asp Pro Thr Asp

65 70 75 80

Gly Ser Ile Leu Ala Cys Asp Pro Gly Leu Ser Arg Thr Cys Asp Gln

85 90 95

Asn Thr Tyr Leu Ser Gly Leu Cys Tyr Leu Phe Arg Gln Asn Leu Gln

100 105 110

Gly Pro Met Leu Gln Gly Arg Pro Gly Phe Gln Glu Cys Ile Lys Gly

115 120 125

Asn Val Asp Leu Val Phe Leu Phe Asp Gly Ser Met Ser Leu Gln Pro

130 135 140

Asp Glu Phe Gln Lys Ile Leu Asp Phe Met Lys Asp Val Met Lys Lys

145 150 155 160

Leu Ser Asn Thr Ser Tyr Gln Phe Ala Ala Val Gln Phe Ser Thr Ser

165 170 175

Tyr Lys Thr Glu Phe Asp Phe Ser Asp Tyr Val Lys Arg Lys Asp Pro

180 185 190

Asp Ala Leu Leu Lys His Val Lys His Met Leu Leu Leu Thr Asn Thr

195 200 205

Phe Gly Ala Ile Asn Tyr Val Ala Thr Glu Val Phe Arg Glu Glu Leu

210 215 220

Gly Ala Arg Pro Asp Ala Thr Lys Val Leu Ile Ile Ile Thr Asp Gly

225 230 235 240

Glu Ala Thr Asp Ser Gly Asn Ile Asp Ala Ala Lys Asp Ile Ile Arg

245 250 255

Tyr Ile Ile Gly Ile Gly Lys His Phe Gln Thr Lys Glu Ser Gln Glu

260 265 270

Thr Leu His Lys Phe Ala Ser Lys Pro Ala Ser Glu Phe Val Lys Ile

275 280 285

Leu Asp Thr Phe Glu Lys Leu Lys Asp Leu Phe Thr Glu Leu Gln Lys

290 295 300

Lys Ile Tyr Val Ile Glu Gly Thr Ser Lys Gln Asp Leu Thr Ser Phe

305 310 315 320

Asn Met Glu Leu Ser Ser Ser Gly Ile Ser Ala Asp Leu Ser Arg Gly

325 330 335

His Ala Val Val Gly Ala Val Gly Ala Lys Asp Trp Ala Gly Gly Phe

340 345 350

Leu Asp Leu Lys Ala Asp Leu Gln Asp Asp Thr Phe Ile Gly Asn Glu

355 360 365

Pro Leu Thr Pro Glu Val Arg Ala Gly Tyr Leu Gly Tyr Thr Val Thr

370 375 380

Trp Leu Pro Ser Arg Gln Lys Thr Ser Leu Leu Ala Ser Gly Ala Pro

385 390 395 400

Arg Tyr Gln His Met Gly Arg Val Leu Leu Phe Gln Glu Pro Gln Gly

405 410 415

Gly Gly His Trp Ser Gln Val Gln Thr Ile His Gly Thr Gln Ile Gly

420 425 430

Ser Tyr Phe Gly Gly Glu Leu Cys Gly Val Asp Val Asp Gln Asp Gly

435 440 445

Glu Thr Glu Leu Leu Leu Ile Gly Ala Pro Leu Phe Tyr Gly Glu Gln

450 455 460

Arg Gly Gly Arg Val Phe Ile Tyr Gln Arg Arg Gln Leu Gly Phe Glu

465 470 475 480

Glu Val Ser Glu Leu Gln Gly Asp Pro Gly Tyr Pro Leu Gly Arg Phe

485 490 495

Gly Glu Ala Ile Thr Ala Leu Thr Asp Ile Asn Gly Asp Gly Leu Val

500 505 510

Asp Val Ala Val Gly Ala Pro Leu Glu Glu Gln Gly Ala Val Tyr Ile

515 520 525

Phe Asn Gly Arg His Gly Gly Leu Ser Pro Gln Pro Ser Gln Arg Ile

530 535 540

Glu Gly Thr Gln Val Leu Ser Gly Ile Gln Trp Phe Gly Arg Ser Ile

545 550 555 560

His Gly Val Lys Asp Leu Glu Gly Asp Gly Leu Ala Asp Val Ala Val

565 570 575

Gly Ala Glu Ser Gln Met Ile Val Leu Ser Ser Arg Pro Val Val Asp

580 585 590

Met Val Thr Leu Met Ser Phe Ser Pro Ala Glu Ile Pro Val His Glu

595 600 605

Val Glu Cys Ser Tyr Ser Thr Ser Asn Lys Met Lys Glu Gly Val Asn

610 615 620

Ile Thr Ile Cys Phe Gln Ile Lys Ser Leu Ile Pro Gln Phe Gln Gly

625 630 635 640

Arg Leu Val Ala Asn Leu Thr Tyr Thr Leu Gln Leu Asp Gly His Arg

645 650 655

Thr Arg Arg Arg Gly Leu Phe Pro Gly Gly Arg His Glu Leu Arg Arg

660 665 670

Asn Ile Ala Val Thr Thr Ser Met Ser Cys Thr Asp Phe Ser Phe His

675 680 685

Phe Pro Val Cys Val Gln Asp Leu Ile Ser Pro Ile Asn Val Ser Leu

690 695 700

Asn Phe Ser Leu Trp Glu Glu Glu Gly Thr Pro Arg Asp Gln Arg Ala

705 710 715 720

Gln Gly Lys Asp Ile Pro Pro Ile Leu Arg Pro Ser Leu His Ser Glu

725 730 735

Thr Trp Glu Ile Pro Phe Glu Lys Asn Cys Gly Glu Asp Lys Lys Cys

740 745 750

Glu Ala Asn Leu Arg Val Ser Phe Ser Pro Ala Arg Ser Arg Ala Leu

755 760 765

Arg Leu Thr Ala Phe Ala Ser Leu Ser Val Glu Leu Ser Leu Ser Asn

770 775 780

Leu Glu Glu Asp Ala Tyr Trp Val Gln Leu Asp Leu His Phe Pro Pro

785 790 795 800

Gly Leu Ser Phe Arg Lys Val Glu Met Leu Lys Pro His Ser Gln Ile

805 810 815

Pro Val Ser Cys Glu Glu Leu Pro Glu Glu Ser Arg Leu Leu Ser Arg

820 825 830

Ala Leu Ser Cys Asn Val Ser Ser Pro Ile Phe Lys Ala Gly His Ser

835 840 845

Val Ala Leu Gln Met Met Phe Asn Thr Leu Val Asn Ser Ser Trp Gly

850 855 860

Asp Ser Val Glu Leu His Ala Asn Val Thr Cys Asn Asn Glu Asp Ser

865 870 875 880

Asp Leu Leu Glu Asp Asn Ser Ala Thr Thr Ile Ile Pro Ile Leu Tyr

885 890 895

Pro Ile Asn Ile Leu Ile Gln Asp Gln Glu Asp Ser Thr Leu Tyr Val

900 905 910

Ser Phe Thr Pro Lys Gly Pro Lys Ile His Gln Val Lys His Met Tyr

915 920 925

Gln Val Arg Ile Gln Pro Ser Ile His Asp His Asn Ile Pro Thr Leu

930 935 940

Glu Ala Val Val Gly Val Pro Gln Pro Pro Ser Glu Gly Pro Ile Thr

945 950 955 960

His Gln Trp Ser Val Gln Met Glu Pro Pro Val Pro Cys His Tyr Glu

965 970 975

Asp Leu Glu Arg Leu Pro Asp Ala Ala Glu Pro Cys Leu Pro Gly Ala

980 985 990

Leu Phe Arg Cys Pro Val Val Phe Arg Gln Glu Ile Leu Val Gln Val

995 1000 1005

Ile Gly Thr Leu Glu Leu Val Gly Glu Ile Glu Ala Ser Ser Met

1010 1015 1020

Phe Ser Leu Cys Ser Ser Leu Ser Ile Ser Phe Asn Ser Ser Lys

1025 1030 1035

His Phe His Leu Tyr Gly Ser Asn Ala Ser Leu Ala Gln Val Val

1040 1045 1050

Met Lys Val Asp Val Val Tyr Glu Lys Gln Met Leu

1055 1060 1065

<210> 55

<211> 678

<212> PRT

<213> Faux Sequence

<220>

<223> Extracellular domain (ECD) LFA-1beta

<400> 55

Gln Glu Cys Thr Lys Phe Lys Val Ser Ser Cys Arg Glu Cys Ile Glu

1 5 10 15

Ser Gly Pro Gly Cys Thr Trp Cys Gln Lys Leu Asn Phe Thr Gly Pro

20 25 30

Gly Asp Pro Asp Ser Ile Arg Cys Asp Thr Arg Pro Gln Leu Leu Met

35 40 45

Arg Gly Cys Ala Ala Asp Asp Ile Met Asp Pro Thr Ser Leu Ala Glu

50 55 60

Thr Gln Glu Asp His Asn Gly Gly Gln Lys Gln Leu Ser Pro Gln Lys

65 70 75 80

Val Thr Leu Tyr Leu Arg Pro Gly Gln Ala Ala Ala Phe Asn Val Thr

85 90 95

Phe Arg Arg Ala Lys Gly Tyr Pro Ile Asp Leu Tyr Tyr Leu Met Asp

100 105 110

Leu Ser Tyr Ser Met Leu Asp Asp Leu Arg Asn Val Lys Lys Leu Gly

115 120 125

Gly Asp Leu Leu Arg Ala Leu Asn Glu Ile Thr Glu Ser Gly Arg Ile

130 135 140

Gly Phe Gly Ser Phe Val Asp Lys Thr Val Leu Pro Phe Val Asn Thr

145 150 155 160

His Pro Asp Lys Leu Arg Asn Pro Cys Pro Asn Lys Glu Lys Glu Cys

165 170 175

Gln Pro Pro Phe Ala Phe Arg His Val Leu Lys Leu Thr Asn Asn Ser

180 185 190

Asn Gln Phe Gln Thr Glu Val Gly Lys Gln Leu Ile Ser Gly Asn Leu

195 200 205

Asp Ala Pro Glu Gly Gly Leu Asp Ala Met Met Gln Val Ala Ala Cys

210 215 220

Pro Glu Glu Ile Gly Trp Arg Asn Val Thr Arg Leu Leu Val Phe Ala

225 230 235 240

Thr Asp Asp Gly Phe His Phe Ala Gly Asp Gly Lys Leu Gly Ala Ile

245 250 255

Leu Thr Pro Asn Asp Gly Arg Cys His Leu Glu Asp Asn Leu Tyr Lys

260 265 270

Arg Ser Asn Glu Phe Asp Tyr Pro Ser Val Gly Gln Leu Ala His Lys

275 280 285

Leu Ala Glu Asn Asn Ile Gln Pro Ile Phe Ala Val Thr Ser Arg Met

290 295 300

Val Lys Thr Tyr Glu Lys Leu Thr Glu Ile Ile Pro Lys Ser Ala Val

305 310 315 320

Gly Glu Leu Ser Glu Asp Ser Ser Asn Val Val Gln Leu Ile Lys Asn

325 330 335

Ala Tyr Asn Lys Leu Ser Ser Arg Val Phe Leu Asp His Asn Ala Leu

340 345 350

Pro Asp Thr Leu Lys Val Thr Tyr Asp Ser Phe Cys Ser Asn Gly Val

355 360 365

Thr His Arg Asn Gln Pro Arg Gly Asp Cys Asp Gly Val Gln Ile Asn

370 375 380

Val Pro Ile Thr Phe Gln Val Lys Val Thr Ala Thr Glu Cys Ile Gln

385 390 395 400

Glu Gln Ser Phe Val Ile Arg Ala Leu Gly Phe Thr Asp Ile Val Thr

405 410 415

Val Gln Val Leu Pro Gln Cys Glu Cys Arg Cys Arg Asp Gln Ser Arg

420 425 430

Asp Arg Ser Leu Cys His Gly Lys Gly Phe Leu Glu Cys Gly Ile Cys

435 440 445

Arg Cys Asp Thr Gly Tyr Ile Gly Lys Asn Cys Glu Cys Gln Thr Gln

450 455 460

Gly Arg Ser Ser Gln Glu Leu Glu Gly Ser Cys Arg Lys Asp Asn Asn

465 470 475 480

Ser Ile Ile Cys Ser Gly Leu Gly Asp Cys Val Cys Gly Gln Cys Leu

485 490 495

Cys His Thr Ser Asp Val Pro Gly Lys Leu Ile Tyr Gly Gln Tyr Cys

500 505 510

Glu Cys Asp Thr Ile Asn Cys Glu Arg Tyr Asn Gly Gln Val Cys Gly

515 520 525

Gly Pro Gly Arg Gly Leu Cys Phe Cys Gly Lys Cys Arg Cys His Pro

530 535 540

Gly Phe Glu Gly Ser Ala Cys Gln Cys Glu Arg Thr Thr Glu Gly Cys

545 550 555 560

Leu Asn Pro Arg Arg Val Glu Cys Ser Gly Arg Gly Arg Cys Arg Cys

565 570 575

Asn Val Cys Glu Cys His Ser Gly Tyr Gln Leu Pro Leu Cys Gln Glu

580 585 590

Cys Pro Gly Cys Pro Ser Pro Cys Gly Lys Tyr Ile Ser Cys Ala Glu

595 600 605

Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly Lys Asn Cys Ser Ala Ala

610 615 620

Cys Pro Gly Leu Gln Leu Ser Asn Asn Pro Val Lys Gly Arg Thr Cys

625 630 635 640

Lys Glu Arg Asp Ser Glu Gly Cys Trp Val Ala Tyr Thr Leu Glu Gln

645 650 655

Gln Asp Gly Met Asp Arg Tyr Leu Ile Tyr Val Asp Glu Ser Arg Glu

660 665 670

Cys Val Ala Gly Pro Asn

675

<210> 56

<211> 674

<212> PRT

<213> Faux Sequence

<220>

<223> Extracellular domain (ECD) VCAM-1

<400> 56

Phe Lys Ile Glu Thr Thr Pro Glu Ser Arg Tyr Leu Ala Gln Ile Gly

1 5 10 15

Asp Ser Val Ser Leu Thr Cys Ser Thr Thr Gly Cys Glu Ser Pro Phe

20 25 30

Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Asn Gly Lys Val Thr

35 40 45

Asn Glu Gly Thr Thr Ser Thr Leu Thr Met Asn Pro Val Ser Phe Gly

50 55 60

Asn Glu His Ser Tyr Leu Cys Thr Ala Thr Cys Glu Ser Arg Lys Leu

65 70 75 80

Glu Lys Gly Ile Gln Val Glu Ile Tyr Ser Phe Pro Lys Asp Pro Glu

85 90 95

Ile His Leu Ser Gly Pro Leu Glu Ala Gly Lys Pro Ile Thr Val Lys

100 105 110

Cys Ser Val Ala Asp Val Tyr Pro Phe Asp Arg Leu Glu Ile Asp Leu

115 120 125

Leu Lys Gly Asp His Leu Met Lys Ser Gln Glu Phe Leu Glu Asp Ala

130 135 140

Asp Arg Lys Ser Leu Glu Thr Lys Ser Leu Glu Val Thr Phe Thr Pro

145 150 155 160

Val Ile Glu Asp Ile Gly Lys Val Leu Val Cys Arg Ala Lys Leu His

165 170 175

Ile Asp Glu Met Asp Ser Val Pro Thr Val Arg Gln Ala Val Lys Glu

180 185 190

Leu Gln Val Tyr Ile Ser Pro Lys Asn Thr Val Ile Ser Val Asn Pro

195 200 205

Ser Thr Lys Leu Gln Glu Gly Gly Ser Val Thr Met Thr Cys Ser Ser

210 215 220

Glu Gly Leu Pro Ala Pro Glu Ile Phe Trp Ser Lys Lys Leu Asp Asn

225 230 235 240

Gly Asn Leu Gln His Leu Ser Gly Asn Ala Thr Leu Thr Leu Ile Ala

245 250 255

Met Arg Met Glu Asp Ser Gly Ile Tyr Val Cys Glu Gly Val Asn Leu

260 265 270

Ile Gly Lys Asn Arg Lys Glu Val Glu Leu Ile Val Gln Glu Lys Pro

275 280 285

Phe Thr Val Glu Ile Ser Pro Gly Pro Arg Ile Ala Ala Gln Ile Gly

290 295 300

Asp Ser Val Met Leu Thr Cys Ser Val Met Gly Cys Glu Ser Pro Ser

305 310 315 320

Phe Ser Trp Arg Thr Gln Ile Asp Ser Pro Leu Ser Gly Lys Val Arg

325 330 335

Ser Glu Gly Thr Asn Ser Thr Leu Thr Leu Ser Pro Val Ser Phe Glu

340 345 350

Asn Glu His Ser Tyr Leu Cys Thr Val Thr Cys Gly His Lys Lys Leu

355 360 365

Glu Lys Gly Ile Gln Val Glu Leu Tyr Ser Phe Pro Arg Asp Pro Glu

370 375 380

Ile Glu Met Ser Gly Gly Leu Val Asn Gly Ser Ser Val Thr Val Ser

385 390 395 400

Cys Lys Val Pro Ser Val Tyr Pro Leu Asp Arg Leu Glu Ile Glu Leu

405 410 415

Leu Lys Gly Glu Thr Ile Leu Glu Asn Ile Glu Phe Leu Glu Asp Thr

420 425 430

Asp Met Lys Ser Leu Glu Asn Lys Ser Leu Glu Met Thr Phe Ile Pro

435 440 445

Thr Ile Glu Asp Thr Gly Lys Ala Leu Val Cys Gln Ala Lys Leu His

450 455 460

Ile Asp Asp Met Glu Phe Glu Pro Lys Gln Arg Gln Ser Thr Gln Thr

465 470 475 480

Leu Tyr Val Asn Val Ala Pro Arg Asp Thr Thr Val Leu Val Ser Pro

485 490 495

Ser Ser Ile Leu Glu Glu Gly Ser Ser Val Asn Met Thr Cys Leu Ser

500 505 510

Gln Gly Phe Pro Ala Pro Lys Ile Leu Trp Ser Arg Gln Leu Pro Asn

515 520 525

Gly Glu Leu Gln Pro Leu Ser Glu Asn Ala Thr Leu Thr Leu Ile Ser

530 535 540

Thr Lys Met Glu Asp Ser Gly Val Tyr Leu Cys Glu Gly Ile Asn Gln

545 550 555 560

Ala Gly Arg Ser Arg Lys Glu Val Glu Leu Ile Ile Gln Val Thr Pro

565 570 575

Lys Asp Ile Lys Leu Thr Ala Phe Pro Ser Glu Ser Val Lys Glu Gly

580 585 590

Asp Thr Val Ile Ile Ser Cys Thr Cys Gly Asn Val Pro Glu Thr Trp

595 600 605

Ile Ile Leu Lys Lys Lys Ala Glu Thr Gly Asp Thr Val Leu Lys Ser

610 615 620

Ile Asp Gly Ala Tyr Thr Ile Arg Lys Ala Gln Leu Lys Asp Ala Gly

625 630 635 640

Val Tyr Glu Cys Glu Ser Lys Asn Lys Val Gly Ser Gln Leu Arg Ser

645 650 655

Leu Thr Leu Asp Val Gln Gly Arg Glu Asn Asn Lys Asp Tyr Phe Ser

660 665 670

ProGlu

<210> 57

<211> 294

<212> PRT

<213> Faux Sequence

<220>

<223> Extracellular domain (ECD) of L-selectin

<400> 57

Trp Thr Tyr His Tyr Ser Glu Lys Pro Met Asn Trp Gln Arg Ala Arg

1 5 10 15

Arg Phe Cys Arg Asp Asn Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys

20 25 30

Ala Glu Ile Glu Tyr Leu Glu Lys Thr Leu Pro Phe Ser Arg Ser Tyr

35 40 45

Tyr Trp Ile Gly Ile Arg Lys Ile Gly Gly Ile Trp Thr Trp Val Gly

50 55 60

Thr Asn Lys Ser Leu Thr Glu Glu Ala Glu Asn Trp Gly Asp Gly Glu

65 70 75 80

Pro Asn Asn Lys Lys Asn Lys Glu Asp Cys Val Glu Ile Tyr Ile Lys

85 90 95

Arg Asn Lys Asp Ala Gly Lys Trp Asn Asp Asp Ala Cys His Lys Leu

100 105 110

Lys Ala Ala Leu Cys Tyr Thr Ala Ser Cys Gln Pro Trp Ser Cys Ser

115 120 125

Gly His Gly Glu Cys Val Glu Ile Ile Asn Asn Tyr Thr Cys Asn Cys

130 135 140

Asp Val Gly Tyr Tyr Gly Pro Gln Cys Gln Phe Val Ile Gln Cys Glu

145 150 155 160

Pro Leu Glu Ala Pro Glu Leu Gly Thr Met Asp Cys Thr His Pro Leu

165 170 175

Gly Asn Phe Ser Phe Ser Ser Gln Cys Ala Phe Ser Cys Ser Glu Gly

180 185 190

Thr Asn Leu Thr Gly Ile Glu Glu Thr Thr Cys Gly Pro Phe Gly Asn

195 200 205

Trp Ser Ser Pro Glu Pro Thr Cys Gln Val Ile Gln Cys Glu Pro Leu

210 215 220

Ser Ala Pro Asp Leu Gly Ile Met Asn Cys Ser His Pro Leu Ala Ser

225 230 235 240

Phe Ser Phe Thr Ser Ala Cys Thr Phe Ile Cys Ser Glu Gly Thr Glu

245 250 255

Leu Ile Gly Lys Lys Lys Thr Ile Cys Glu Ser Ser Gly Ile Trp Ser

260 265 270

Asn Pro Ser Pro Ile Cys Gln Lys Leu Asp Lys Ser Phe Ser Met Ile

275 280 285

Lys Glu Gly Asp Tyr Asn

290

<210> 58

<211> 943

<212> PRT

<213> Faux Sequence

<220>

<223> Extracellular domain (ECD) VLA-4

<400> 58

Asn Val Asp Thr Glu Ser Ala Leu Leu Tyr Gln Gly Pro His Asn Thr

1 5 10 15

Leu Phe Gly Tyr Ser Val Val Leu His Ser His Gly Ala Asn Arg Trp

20 25 30

Leu Leu Val Gly Ala Pro Thr Ala Asn Trp Leu Ala Asn Ala Ser Val

35 40 45

Ile Asn Pro Gly Ala Ile Tyr Arg Cys Arg Ile Gly Lys Asn Pro Gly

50 55 60

Gln Thr Cys Glu Gln Leu Gln Leu Gly Ser Pro Asn Gly Glu Pro Cys

65 70 75 80

Gly Lys Thr Cys Leu Glu Glu Arg Asp Asn Gln Trp Leu Gly Val Thr

85 90 95

Leu Ser Arg Gln Pro Gly Glu Asn Gly Ser Ile Val Thr Cys Gly His

100 105 110

Arg Trp Lys Asn Ile Phe Tyr Ile Lys Asn Glu Asn Lys Leu Pro Thr

115 120 125

Gly Gly Cys Tyr Gly Val Pro Pro Asp Leu Arg Thr Glu Leu Ser Lys

130 135 140

Arg Ile Ala Pro Cys Tyr Gln Asp Tyr Val Lys Lys Phe Gly Glu Asn

145 150 155 160

Phe Ala Ser Cys Gln Ala Gly Ile Ser Ser Phe Tyr Thr Lys Asp Leu

165 170 175

Ile Val Met Gly Ala Pro Gly Ser Ser Tyr Trp Thr Gly Ser Leu Phe

180 185 190

Val Tyr Asn Ile Thr Asn Lys Tyr Lys Ala Phe Leu Asp Lys Gln

195 200 205

Asn Gln Val Lys Phe Gly Ser Tyr Leu Gly Tyr Ser Val Gly Ala Gly

210 215 220

His Phe Arg Ser Gln His Thr Thr Glu Val Val Gly Gly Ala Pro Gln

225 230 235 240

His Glu Gln Ile Gly Lys Ala Tyr Ile Phe Ser Ile Asp Glu Lys Glu

245 250 255

Leu Asn Ile Leu His Glu Met Lys Gly Lys Lys Leu Gly Ser Tyr Phe

260 265 270

Gly Ala Ser Val Cys Ala Val Asp Leu Asn Ala Asp Gly Phe Ser Asp

275 280 285

Leu Leu Val Gly Ala Pro Met Gln Ser Thr Ile Arg Glu Glu Gly Arg

290 295 300

Val Phe Val Tyr Ile Asn Ser Gly Ser Gly Ala Val Met Asn Ala Met

305 310 315 320

Glu Thr Asn Leu Val Gly Ser Asp Lys Tyr Ala Ala Arg Phe Gly Glu

325 330 335

Ser Ile Val Asn Leu Gly Asp Ile Asp Asn Asp Gly Phe Glu Asp Val

340 345 350

Ala Ile Gly Ala Pro Gln Glu Asp Asp Leu Gln Gly Ala Ile Tyr Ile

355 360 365

Tyr Asn Gly Arg Ala Asp Gly Ile Ser Ser Thr Phe Ser Gln Arg Ile

370 375 380

Glu Gly Leu Gln Ile Ser Lys Ser Leu Ser Met Phe Gly Gln Ser Ile

385 390 395 400

Ser Gly Gln Ile Asp Ala Asp Asn Asn Gly Tyr Val Asp Val Ala Val

405 410 415

Gly Ala Phe Arg Ser Asp Ser Ala Val Leu Leu Arg Thr Arg Pro Val

420 425 430

Val Ile Val Asp Ala Ser Leu Ser His Pro Glu Ser Val Asn Arg Thr

435 440 445

Lys Phe Asp Cys Val Glu Asn Gly Trp Pro Ser Val Cys Ile Asp Leu

450 455 460

Thr Leu Cys Phe Ser Tyr Lys Gly Lys Glu Val Pro Gly Tyr Ile Val

465 470 475 480

Leu Phe Tyr Asn Met Ser Leu Asp Val Asn Arg Lys Ala Glu Ser Pro

485 490 495

Pro Arg Phe Tyr Phe Ser Ser Asn Gly Thr Ser Asp Val Ile Thr Gly

500 505 510

Ser Ile Gln Val Ser Ser Arg Glu Ala Asn Cys Arg Thr His Gln Ala

515 520 525

Phe Met Arg Lys Asp Val Arg Asp Ile Leu Thr Pro Ile Gln Ile Glu

530 535 540

Ala Ala Tyr His Leu Gly Pro His Val Ile Ser Lys Arg Ser Thr Glu

545 550 555 560

Glu Phe Pro Pro Leu Gln Pro Ile Leu Gln Gln Lys Lys Glu Lys Asp

565 570 575

Ile Met Lys Lys Thr Ile Asn Phe Ala Arg Phe Cys Ala His Glu Asn

580 585 590

Cys Ser Ala Asp Leu Gln Val Ser Ala Lys Ile Gly Phe Leu Lys Pro

595 600 605

His Glu Asn Lys Thr Tyr Leu Ala Val Gly Ser Met Lys Thr Leu Met

610 615 620

Leu Asn Val Ser Leu Phe Asn Ala Gly Asp Asp Ala Tyr Glu Thr Thr

625 630 635 640

Leu His Val Lys Leu Pro Val Gly Leu Tyr Phe Ile Lys Ile Leu Glu

645 650 655

Leu Glu Glu Lys Gln Ile Asn Cys Glu Val Thr Asp Asn Ser Gly Val

660 665 670

Val Gln Leu Asp Cys Ser Ile Gly Tyr Ile Tyr Val Asp His Leu Ser

675 680 685

Arg Ile Asp Ile Ser Phe Leu Leu Asp Val Ser Ser Leu Ser Arg Ala

690 695 700

Glu Glu Asp Leu Ser Ile Thr Val His Ala Thr Cys Glu Asn Glu Glu

705 710 715 720

Glu Met Asp Asn Leu Lys His Ser Arg Val Thr Val Ala Ile Pro Leu

725 730 735

Lys Tyr Glu Val Lys Leu Thr Val His Gly Phe Val Asn Pro Thr Ser

740 745 750

Phe Val Tyr Gly Ser Asn Asp Glu Asn Glu Pro Glu Thr Cys Met Val

755 760 765

Glu Lys Met Asn Leu Thr Phe His Val Ile Asn Thr Gly Asn Ser Met

770 775 780

Ala Pro Asn Val Ser Val Glu Ile Met Val Pro Asn Ser Phe Ser Pro

785 790 795 800

Gln Thr Asp Lys Leu Phe Asn Ile Leu Asp Val Gln Thr Thr Thr Gly

805 810 815

Glu Cys His Phe Glu Asn Tyr Gln Arg Val Cys Ala Leu Glu Gln Gln

820 825 830

Lys Ser Ala Met Gln Thr Leu Lys Gly Ile Val Arg Phe Leu Ser Lys

835 840 845

Thr Asp Lys Arg Leu Leu Tyr Cys Ile Lys Ala Asp Pro His Cys Leu

850 855 860

Asn Phe Leu Cys Asn Phe Gly Lys Met Glu Ser Gly Lys Glu Ala Ser

865 870 875 880

Val His Ile Gln Leu Glu Gly Arg Pro Ser Ile Leu Glu Met Asp Glu

885 890 895

Thr Ser Ala Leu Lys Phe Glu Ile Arg Ala Thr Gly Phe Pro Glu Pro

900 905 910

Asn Pro Arg Val Ile Glu Leu Asn Lys Asp Glu Asn Val Ala His Val

915 920 925

Leu Leu Glu Gly Leu His His Gln Arg Pro Lys Arg Tyr Phe Thr

930 935 940

<210> 59

<211> 127

<212> PRT

<213> Streptomyces avidinii

<220>

<221> OTHER SIGN

<223> Minimal streptavidin

<400> 59

Met Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr

1 5 10 15

Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Glu

20 25 30

Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr

35 40 45

Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr

50 55 60

Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp

65 70 75 80

Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp

85 90 95

Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu

100 105 110

Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 60

<211> 127

<212> PRT

<213> Streptomyces avidinii

<220>

<221> OTHER SIGN

<223> Streptavidin mutein Val44-Thr45-Ala46-Arg47

<400> 60

Met Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr

1 5 10 15

Phe Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Val

20 25 30

Thr Ala Arg Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr

35 40 45

Asp Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr

50 55 60

Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp

65 70 75 80

Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp

85 90 95

Leu Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu

100 105 110

Val Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

<210> 61

<211> 126

<212> PRT

<213> Streptomyces avidinii

<220>

<221> OTHER SIGN

<223> Streptavidin mutein Ile44-Gly45-Ala-46-Arg47

<400> 61

Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn Gln Leu Gly Ser Thr Phe

1 5 10 15

Ile Val Thr Ala Gly Ala Asp Gly Ala Leu Thr Gly Thr Tyr Ile Gly

20 25 30

Ala Arg Gly Asn Ala Glu Ser Arg Tyr Val Leu Thr Gly Arg Tyr Asp

35 40 45

Ser Ala Pro Ala Thr Asp Gly Ser Gly Thr Ala Leu Gly Trp Thr Val

50 55 60

Ala Trp Lys Asn Asn Tyr Arg Asn Ala His Ser Ala Thr Thr Trp Ser

65 70 75 80

Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg Ile Asn Thr Gln Trp Leu

85 90 95

Leu Thr Ser Gly Thr Thr Glu Ala Asn Ala Trp Lys Ser Thr Leu Val

100 105 110

Gly His Asp Thr Phe Thr Lys Val Lys Pro Ser Ala Ala Ser

115 120 125

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Claims (90)

1. A particulate oligomeric reagent for multimerization of one or more binding agents, wherein the particulate oligomeric reagent is a soluble oligomer of 1500 to 7500 tetramers of streptavidin or a functionally active fragment thereof or a streptavidin mutein molecule or a functionally active fragment thereof containing the amino acid sequence Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at sequence positions corresponding to positions 44-47 of the amino acid sequence shown in SEQ ID NO: 1. 2. The oligomeric particulate reagent of claim 1, wherein each of the streptavidin or streptavidin mutein molecules or functionally active fragments thereof contains a binding site for biotin, a biotin analog, or a streptavidin-binding peptide. 3. An oligomeric reagent in the form of particles according to claim 1 or 2, where each of the functionally active fragments begins at the N-terminus in the region of amino acid positions 10-16 of the SEQ ID NO: 1 sequence and ends at the C-terminus in the region of amino acid positions 133- 142 sequences SEQ ID NO: 1. 4. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-3, wherein the particulate oligomeric reagent is a soluble oligomer of 1500 to 7500 tetramers of a streptavidin mutein molecule or a functional fragment thereof. 5. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-4, wherein each of the streptavidin mutein molecules or their functionally active fragments reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide. 6. Oligomeric reagent in the form of particles according to any one of paragraphs. 1–5, while each of the streptavidin mutein molecules or their functionally active fragments contains: an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity to any SEQ ID NO: 3-6, 27, 28, 60, or 61, or a functional fragment thereof that reversibly binds to biotin, a biotin analog, or a streptavidin-binding peptide, wherein the amino acid sequence or functional fragment thereof contains the amino acid sequence corresponding to Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ at positions of the sequence corresponding to positions 44-47 of the amino acid sequence represented by SEQ ID NO: 1. 7. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-6, where each of the streptavidin mutein molecules or functionally active fragments thereof contains an amino acid sequence represented by any of SEQ ID NOs: 3-6, 27, 28, 60 or 61, or a functional fragment thereof, which reversibly binds to biotin, an analogue biotin or streptavidin-binding peptide, where its functional fragment contains the amino acid sequence corresponding to Val ⁴⁴ -Thr ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ or Ile ⁴⁴ -Gly ⁴⁵ -Ala ⁴⁶ -Arg ⁴⁷ , in positions of the sequence corresponding to positions 44-47 of the amino acid the sequence shown by SEQ ID NO: 1. 8. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-7, where each of the streptavidin mutein molecules or their functionally active fragments reversibly binds to a streptavidin-binding peptide. 9. Oligomeric reagent in the form of particles according to any one of paragraphs. 2-8, where the streptavidin-binding peptide is the amino acid sequence represented by SEQ ID NO: 7 or 8. 10. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-9, wherein each of the streptavidin mutein molecules or functionally active fragments thereof contains the amino acid sequence represented by any of SEQ ID NOs: 3-6, 27, 28, 60, or 61. 11. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-10, wherein each of the streptavidin mutein molecules or their functionally active fragments contains the amino acid sequence shown in SEQ ID NO: 6. 12. Oligomeric reagent in the form of particles according to any one of paragraphs. 1–11, while the oligomer contains radius between 50 nm and 150 nm, between 75 nm and 125 nm, between 80 nm and 115 nm, or between 90 nm and 110 nm, each inclusive. 13. Oligomeric reagent in the form of particles according to any one of paragraphs. 1–12, while the oligomer contains a radius of 90 nm ± 15 nm or 95 nm ± 20–25 nm. 14. The particulate oligomeric reagent of claim 12 or 13, wherein the radius is the hydrodynamic radius. 15. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-14, wherein the oligomer has a molecular weight between 5×10 ⁷ g/mol and 5×10 ⁸ g/mol, between 1×10 ⁸ g/mol and 5×10 ⁸ g/mol, or between 1×10 ⁸ g /mol and 2×10 ⁸ g/mol. 16. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-15, wherein the oligomer contains between 2000 and 5000, between 2000 and 3000, or between 2000 and 2500 tetramers. 17. Oligomeric reagent in the form of particles according to any one of paragraphs. 1–16, while less than 20%, 10%, 5%, 1% of the lysine residues of the oligomer contain N-substituted iminothiolane. 18. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-17, wherein the streptavidin or streptavidin mutein molecules or functionally active oligomer fragments thereof are crosslinked with a heterobifunctional crosslinker. 19. Oligomeric reagent in the form of particles according to any one of paragraphs. 1-18, where streptavidin or streptavidin mutein molecules or their functionally active fragments of the oligomer are crosslinked by a crosslinking agent for amine and thiol groups. 20. Multimerized oligomeric reagent in the form of particles for stimulating cells, containing oligomeric reagent in the form of particles according to any one of paragraphs. 1-19, the first receptor-binding agent and the second receptor-binding agent, where: the first receptor-binding agent comprises (i) a first binding partner that is associated with one or more first binding sites of streptavidin or streptavidin mutein molecules or functionally active particulate oligomeric reagent fragments thereof, and (ii) a first antibody or antibody fragment that binds to a receptor expressed on the surface of the target cell to induce a primary activation signal in the target cell; and the second receptor-binding agent comprises (i) a second binding partner that is associated with one or more second binding sites of streptavidin or streptavidin mutein molecules or their functionally active particulate oligomeric reagent fragments, and (ii) a second antibody or antibody fragment that binds to a costimulatory molecule expressed on the surface of a target cell to induce a costimulatory signal in the target cell. 21. The multimerized particulate oligomeric reagent of claim 20, wherein the particulate oligomeric reagent is a soluble oligomer of between 1500 and 7500 tetramers of the streptavidin mutein molecule or a functional fragment thereof. 22. The multimerized oligomeric particulate reagent of claim 20 or 21, wherein each of the streptavidin mutein molecules or functionally active fragments thereof contains an amino acid sequence represented by any of SEQ ID NOs: 3-6, 27, 28, 60, or 61. 23. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-22, where each of the streptavidin mutein molecules or their functionally active fragments contains the amino acid sequence represented by SEQ ID NO: 6. 24. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-23, where: the first binding partner is reversibly linked to one or more first binding sites; and/or the second binding partner is reversibly linked to one or more second binding sites. 25. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-24, wherein the first and/or second binding partner comprises biotin, a biotin analog, or a streptavidin-binding peptide. 26. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-25, wherein the first and/or second binding partner contains a streptavidin-binding peptide. 27. The multimerized oligomeric particulate reagent of claim 25 or 26 wherein the streptavidin-binding peptide of the first and/or second binding partner is the amino acid sequence represented by any of SEQ ID NOs: 7, 8 and 15-19. 28. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 25-27, wherein the streptavidin-binding peptide of the first and/or second binding partner is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16). 29. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-28, where the target cell is an immune cell. 30. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-29, where the target cell is a T cell. 31. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-30, where the primary activation signal is a TCR/CD3 complex-associated signal. 32. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-31, where the first receptor binding agent binds to a member of the TCR/CD3 complex. 33. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-32, where the first receptor-binding agent specifically binds to CD3. 34. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-33, wherein the costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. 35. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-34, where the costimulatory molecule is CD28. 36. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 25-35, wherein the first receptor-binding agent comprises a streptavidin-binding peptide and an anti-CD3 antibody or antibody fragment, and the second receptor-binding agent comprises a streptavidin-binding peptide and an anti-CD28 antibody or antibody fragment. 37. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 20-36, where: where the first receptor-binding agent contains the first antibody fragment that binds to the receptor; and/or where the second receptor-binding agent contains the second fragment of the antithede, which binds to the co-stimulatory molecule. 38. The multimerized oligomeric particulate reagent of claim 37, wherein the first and/or second antibody fragment is a monovalent antibody fragment. 39. The multimerized oligomeric particulate reagent of claim 37 or 38, wherein the first and/or second antibody fragment is a Fab. 40. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 25-39, wherein the first receptor-binding agent contains a streptavidin-binding peptide and an anti-CD3 Fab, and the second receptor-binding agent contains a streptavidin-binding peptide and an anti-CD28 Fab. 41. Multimerized oligomeric reagent in the form of particles for cell selection, containing oligomeric reagent in the form of particles according to any one of paragraphs. 1-19 and a selective agent, wherein the selective agent comprises (i) a binding partner that is associated with one or more binding sites of streptavidin or streptavidin mutein molecules or functionally active particulate oligomeric reagent fragments thereof, and (ii) an antibody or antibody fragment , which binds to a selectable marker expressed on the surface of the target cell. 42. The multimerized particulate oligomeric reagent of claim 41, wherein the particulate oligomeric reagent is a soluble oligomer of between 1500 and 7500 tetramers of the streptavidin mutein molecule or a functional fragment thereof. 43. The multimerized oligomeric particulate reagent of claim 41 or 42, wherein each of the streptavidin mutein molecules or functionally active fragments thereof contains an amino acid sequence represented by any of SEQ ID NOs: 3-6, 27, 28, 60, or 61. 44. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-43, each of the streptavidin mutein molecules or their functionally active fragments contains the amino acid sequence represented by SEQ ID NO: 6. 45. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-44 wherein the binding partner is reversibly linked to one or more binding sites. 46. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-45, wherein the binding partner contains biotin, a biotin analog, or a streptavidin-binding peptide. 47. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-46, where the binding partner contains a streptavidin-binding peptide. 48. The multimerized oligomeric particulate reagent of claim 46 or 47 wherein the streptavidin binding peptide is the amino acid sequence represented by any of SEQ ID NOs: 7, 8 and 15-19. 49. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 46-48, where the streptavidin-binding peptide is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16). 50. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-49, where the selective agent contains an antibody fragment that binds to the selectable marker. 51. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-50, where the antibody fragment is a fragment of a monovalent antibody. 52. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-51, where the antibody fragment is a Fab. 53. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-52, where the target cell is an immune cell. 54. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-53, where the target cell is a T cell. 55. Multimerized oligomeric reagent in the form of particles according to any one of paragraphs. 41-54, where the selectable marker is CCR7, CD3, CD4, CD8, CD25, CD28, CD27, CD45RA, CD45RO, CD62L, or CD127. 56. A method for stimulating cells, the method comprising incubating a cell composition containing target cells in the presence of a multimerized oligomeric particulate reagent according to any one of paragraphs. 20–40, thus stimulating target cells. 57. The method of claim 56, wherein the multimerized oligomeric particulate reagent is a soluble oligomer of between 1500 and 7500 tetramers of the streptavidin mutein molecule or a functional fragment thereof. 58. The method according to p. 56 or 57, where each of the streptavidin mutein molecules or their functionally active fragments contains the amino acid sequence represented by any of SEQ ID NO: 3-6, 27, 28, 60 or 61. 59. The method according to any one of paragraphs. 56-58, where each of the streptavidin mutein molecules or their functionally active fragments contains the amino acid sequence represented by SEQ ID NO: 6. 60. The method according to any one of paragraphs. 56-59, wherein the first binding partner is reversibly linked to one or more first binding sites; and/or the second binding partner is reversibly linked to one or more second binding sites. 61. The method according to any one of paragraphs. 56-60, wherein the first and/or second binding partner contains a streptavidin-binding peptide. 62. The method of claim 61 wherein the streptavidin-binding peptide of the first and/or second binding partner is an amino acid sequence represented by any of SEQ ID NOs: 7, 8, and 15-19. 63. The method of claim 61 or 62, wherein the streptavidin-binding peptide of the first and/or second binding partner is SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 16). 64. The method according to any one of paragraphs. 56-63, where: the first receptor-binding agent contains the first antibody fragment that binds to the receptor; and/or the second receptor-binding agent contains a second antibody fragment that binds to a costimulatory molecule. 65. The method of claim 64, wherein the first and/or second antibody fragment is a monovalent antibody fragment. 66. The method of claim 64 or 65, wherein the first and/or second antibody fragment is a Fab. 67. The method according to any one of paragraphs. 56–66, with the target cells being immune cells. 68. The method according to any one of paragraphs. 56–67, with the target cells being T cells. 69. The method according to any one of paragraphs. 56–68, with the primary activation signal being a signal associated with the TCR/CD3 complex. 70. The method according to any one of paragraphs. 56-69, where the first receptor-binding agent binds to a member of the TCR/CD3 complex. 71. The method according to any one of paragraphs. 56-70, where the first receptor-binding agent specifically binds to CD3. 72. The method according to any one of paragraphs. 56-71, where the costimulatory molecule is CD28, CD90 (Thy-1), CD95 (Apo-/Fas), CD137 (4-1BB), CD154 (CD40L), ICOS, LAT, CD27, OX40, or HVEM. 73. The method according to any one of paragraphs. 56-72, wherein the costimulatory molecule is CD28. 74. The method according to any one of paragraphs. 61-63 and 67-73, wherein the first receptor-binding agent comprises a streptavidin-binding peptide and an anti-CD3 antibody or antibody fragment, and the second receptor-binding agent comprises a streptavidin-binding peptide and an anti-CD28 antibody or antibody fragment. 75. The method according to any one of paragraphs. 61-74, wherein the first receptor-binding agent contains a streptavidin-binding peptide and an anti-CD3 Fab, and the second receptor-binding agent contains a streptavidin-binding peptide and an anti-CD28 Fab. 76. The method according to any one of paragraphs. 56-75, while the target cells express the recombinant receptor. 77. The method of claim 76 wherein the recombinant receptor is a recombinant T cell receptor or chimeric antigen receptor (CAR). 78. The method according to any one of paragraphs. 60-77, further comprising breaking the reversible bond between the first binding partner and one or more first binding sites and/or between the second binding partner and one or more second binding sites, said breaking comprising introducing into the target cells a substance, capable of competing for binding with one or more first binding sites and/or one or more second binding sites. 79. The method of claim 78, wherein the substance contains biotin or a biotin analogue. 80. The method according to item 78 or 79, while the substance contains D-biotin.

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