WO2024255853A1

WO2024255853A1 - Cell-based assays using immobilized recombinant proteins to measure immune regulation of therapeutic biologics

Info

Publication number: WO2024255853A1
Application number: PCT/CN2024/099248
Authority: WO
Inventors: Xiaoqing JIA; Min Chen
Original assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd; Wuxi Biologics Ireland Ltd
Priority date: 2023-06-16
Filing date: 2024-06-14
Publication date: 2024-12-19
Anticipated expiration: 2025-12-16

Abstract

Provided herein are cell-based assays using immobilized recombinant proteins to measure immune regulation of therapeutic biologics. In particular, the disclosure relates to a method for assessing and/or determining immune response induced by a biologic molecule comprising: a) immobilizing a recombinant membrane protein or a fragment comprising the extracellular domain thereof on a support; b) contacting the immobilized protein or fragment with (i) a biologic molecule (such as an antibody); and (ii) an effector cell; and c) detecting the activation of the effector cell so as to assess and/or determine the immune response (such as ADCC, ADCP) induced by the biologic molecule.

Description

Cell-Based Assays Using Immobilized Recombinant Proteins to Measure Immune Regulation of Therapeutic Biologics

TECHNICAL FIELD

The present disclosure concerns the field of bio-pharm analytics and clinical diagnosis and therapy. It inter alia pertains to innovative cell-based assays using immobilized recombinant proteins to measure immune regulation of therapeutic biologics.

BACKGROUND

Membrane proteins anchor to cell membranes and have functions and mechanism of actions different from soluble proteins. These membrane proteins form selectively permeable barriers that control the flow of molecules into and out of cells. They also act as receptors that bind to specific molecules and transporters that move molecules across membranes. Membrane proteins are critical for cellular communication and signaling, metabolism and immune response ^[1～2] .

By way of example, membrane proteins are pivotal for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) . ADCC and ADCP are antibody effector functions mediated through (a) antibody binding to membrane proteins expressed on the target cells, and (b) engagement of Fcγ receptors (FcγRs) expressed on effector cells (e.g. natural killer (NK) cells or myeloid cells) ^[3～4] . For ADCC, activated NK cells can release cytotoxic granules (e.g. perforin and granzymes) into target cells, leading to cell death. For ADCP, FcγR engagement results in cytoskeletal rearrangements, phagosome formation and the endocytosis, and degradation of the target cells by lysosomal enzymes presented in the effector cells.

Due to the importance of ADCC and ADCP (also Antibody-dependent trogocytosis (ADPC) ) in therapeutic antibody clinical effects, many in vitro bioassays have been developed to assess and/or determine the efficacy of antibodies in eliciting ADCC and/or ADCP. Among these methods, both FcγR-expressing effector cells (e.g. PBMC or engineered cells) and target cells (e.g. tumor cell lines) are used. These in vitro methods require two cell lines, are labor-intensive and come with intrinsic variability, hence being unreliable in predicting in vivo drug efficacy ^[5] .

Hence, there is a great need for bioassays for measuring immune regulation of therapeutic biologics and the efficacy of therapeutic antibodies in eliciting ADCC and/or ADCP.

SUMMARY OF THE INVENTION

Here, we disclose in vitro bioassays enabling the effective assessment of immune regulation effect (such as ADCC, ADTC and ADCP) of the therapeutic biologics and the control mechanism thereof.

According to one aspect, disclosed herein is a method for assessing and/or determining immune response induced by a biologic molecule comprising:

a) immobilizing a recombinant membrane protein or a fragment comprising the extracellular domain thereof on a support;

b) contacting the immobilized protein or fragment with (i) a biologic molecule; and (ii) an effector cell, wherein the biologic molecule is capable or suspected to be capable of directly or indirectly binding to the immobilized protein or fragment and the effector cell, and activating the effector cell;

c) detecting the activation of the effector cell so as to assess and/or determine the immune response induced by the biologic molecule.

According to one aspect, disclosed herein is a method for assessing and/or determining antibody-dependent cell-mediated immune response, comprising:

a) immobilizing a recombinant membrane protein or an antibody binding fragment thereof on a support;

b) contacting the immobilized protein or fragment with (i) an antibody or a functional fragment thereof (such as a fragment comprising membrane protein binding domain and/or an Fc-containing fragment) ; and (ii) an effector cell;

c) detecting the activation of the effector cell so as to assess and/or determine the antibody-dependent cell-mediated immune response induced by the antibody or the fragment thereof.

In some embodiments, the method can be used for but not limited to:

(a') screening a candidate biologic molecule based on the ability to induce immune response against a target cell expressing the membrane protein or an antibody binding fragment thereof;

(b') controlling the quality of the biologic molecule, during the manufacture, storage, transportation and/or application of the antibody or fragment thereof;

(c') screening a candidate compound for the ability to modulate immune response, wherein the method further comprises adding the candidate compound into the system of step b) ;

(d') optimizing the types and/or concentration of the biologic molecule in inducing antibody-dependent cell-mediated immune response;

(e') predicting the effect of the biologic molecule in treatment of target cell associated diseases; and/or

(f') assessing the interactions between two or more biologic molecules in immune response, wherein the two or more biologic molecule are used in step b) ; and/or

(g') evaluating the immune regulation ability of the membrane proteins or the fragment thereof to the immune response induced by the biologic molecule.

In one aspect, provided herein is a product (such as a system or a kit) for assessing and/or determining immune response, wherein the product comprises means and/or substances for carrying out the method of the present application.

In one aspect, provided herein is a use of means and/or substance (s) for carrying out the method of the present application in the preparation of a product for assessing and/or determining immune response (such as ADCC, ADTC and/or ADCP) .

In one aspect, provided herein is a use of means and/or substance (s) for carrying out the method of the present application in the preparation of a product for screening the biological molecules, controlling the quality of the biological molecules, optimizing the types and/or concentration of the biological molecules, predicting the effect of the biological molecules, assessing the interactions between two or more biologic molecules in immune response, and/or evaluating the immune regulation ability of the membrane proteins or the fragment thereof.

Other objects, features, advantages and aspects of the present application will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following.

BRIEF DESCRTPTION OF THE DRAWINGS

The novel and innovative features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Figure 1: ADCC elicited by immobilized recombinant proteins and effector cell line.

Figure 2: ADCC elicited by 2 cell lines: target and effector cell lines.

Figure 3: Immobilized recombinant proteins allow accurate adjustment of immune stimulation.

Figure 4: ADCP elicited by immobilized recombinant proteins and effector cell line.

Figure 5: ADCP elicited by 2 cell lines: target and effector cell lines.

Figure 6: Immobilized recombinant proteins allow accurate adjustment of immune stimulation.

In Figures 3 and 6:

-X-axis: CD20/HER2/RBD Conc. (μg/mL) : corresponding coating concentrations of immobilized proteins.

-Plot#1 (Rituxan (1 μg/mL) ) : Coating concentrations of immobilized CD20 is 0-8 μg/mL, Rituxan concentration is constantly at 1 μg/mL.

-Plot#2 (Herceptin (1 μg/mL) ) : Coating concentrations of immobilized HER2 is 0-8 μg/mL, Herceptin concentration is constantly at 1 μg/mL.

-Plot#3 (SA2-S36 NAb (1 μg/mL) ) : Coating concentrations of immobilized RBD is 0-8 μg/mL, SA2-S36 Neutralizing Ab concentration is constantly at 1 μg/mL.

-Plot#4 (Rituxan (0 μg/mL) ) : Coating concentrations of immobilized CD20 is 0-8 μg/mL, Rituxan concentration is constantly at 0 μg/mL.

-Plot#5 (Herceptin (0 μg/mL) ) : Coating concentrations of immobilized HER2 is 0-8 μg/mL, Herceptin concentration is constantly at 0 μg/mL.

-Plot#6 (SA2-S36 NAb (0 μg/mL) ) : Coating concentrations of immobilized RBD is 0-8 μg/mL, SA2-S36 Neutralizing Ab concentration is constantly at 0 μg/mL.

-A, B, C, and D in the formulas are 4 parameters of the logistic fitting curve:

A = zero dose response in Luminescence

B = curvature (slope ratio)

C = EC₅₀ value

D = maximum dose response in Luminescence.

DETAILED DESCRIPTION OF THE DISCLOSURE

The following description and examples illustrate embodiments of the invention in detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

This invention innovatively uses the immobilized recombinant protein to simulate the cell membrane surface proteins, and establishes in vitro cell assays to evaluate the immune response inducing functions (such as ADCC and ADCP) of the therapeutic biologics and its control mechanism. The invention has wide application value in the efficacy and safety assessment of biological drugs.

In particular, this is the first report of cell-based immune regulation models using immobilized and highly quantifiable recombinant proteins to simulate the membrane antigens expressed by various target cells (such as tumor or immune cells) . It can be applied to a wide range of cell functional experiments of membrane-expressed proteins and compare the biological functions of free vs. membrane-expressed proteins as related to the assessment of the efficacy and safety of the therapeutic biologics. The results from this assay can be good indicator of changes in biological molecules. Compared to the conventional two-cell line functional assays, this new method is applicable when no natural or engineered target cells are available, it is streamlined with less variability, validatable (such as according to the requirements in ICH Q2 (R1) ) and transferable, hence, a better choice to support biologics product development and manufacturing.

The bioassays invented can be widely used, for example, can be carried out in the analytical labs in the biopharmaceutical company for product quality assessment during product development and manufacturing. They can also be carried out in the clinical labs to measure the clinical responses in a patient.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, preferred methods and materials are described.

As used herein, the term "a" or "an" is intended to mean "one or more" (i.e., at least one) of the grammatical object of the article. Singular expressions, unless defined otherwise in contexts, include plural expressions. By way of example, "an element" means one element or more than one element.

By "about" is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The use of “or” means “and/or” unless stated otherwise.

As used herein, unless otherwise noted, the term "comprise" , "include" and "including" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

The phrase "consisting of" is meant to include, and is limited to, whatever follows the phrase "consisting of. " Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and that other elements may be present.

The term "isolated" refers to a material that is substantially or essentially free from components that normally accompany it in its native state. The material can be a cell or a macromolecule such as a protein or nucleic acid. For example, an "isolated cell, " as used herein, refers to a cell, which has been purified from the cells in a naturally-occurring state.

Recombinant membrane protein, Biological molecule, and effector cell

Provided herein is a novel in vitro system and method for assaying immune response induced by a biological molecule or functional fragment thereof in the presence of a recombinant membrane protein and an effector cell.

As indicated above, the term "a" or "an" is intended to mean "one or more" (i.e., at least one) of the biological molecules or functional fragments thereof, target cells and/or effector cells. Preferably, the system and method can be used in a high throughput assay, such as using a multi-well plate as the support for one or more recombinant membrane proteins in the same or different concentrations and contacting the same with one or more biological molecule or functional fragment thereof in the same or different concentrations and one or more kinds of effector cells in the same or different cell numbers.

As used herein, the term "biological molecule" refers to a molecule capable of binding to a target molecule on a target cell (such as a membrane protein on the surface of a target cell) and an effector cell and inducing desired biological activity (such as inducing ADCC and/or ADCP) . The term "molecule" may refer to a micro-molecule or macro-molecule, such as an antibody or antigen binding fragment thereof, a ligand of a cell surface receptor, a cytokine or a chemokine.

As used herein, the term "antibody or functional antibody fragment thereof" refers to intact molecules as well as to fragments thereof, which are capable of binding to a recombinant membrane protein (such as an antigen, preferably a recombinant membrane protein derived from a membrane protein from the target cell) . By "binding to the recombinant membrane protein" is meant that an antibody is immuno-specific for the recombinant membrane protein, e.g., an antigen on the surface of the target cells. The term "immuno-specific" means that the antibody has substantially greater affinity for the antigen on the target cell than affinity for other proteins (e.g., other related proteins) .

The antibody can be a known antibody that can induce ADCC and/or ADCP or a new or candidate antibody whose effect in inducing ADCC and/or ADCP is unknown and to be determined. The antibody or candidate antibody used in the methods provided herein can be, for example, a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody. Antibodies that can be used in the methods described herein also include antibodies that have been identified as having therapeutic potential (e.g., antibodies that have already undergone clinical trials) .

Any suitable recombinant membrane protein can be selected and used according to the need in practice or to meet the assay of the biological molecule. Preferably, the recombinant membrane protein is capable of being specifically recognized and bound by the antibody or the functional antibody fragment thereof, for example the membrane protein comprises or is an antibody specific antigenic epitope expressed on the surface of a target cell.

In some embodiments, the target cell is a diseased cell or cell line, such as a cancer cell or cell line, an infected cell or cell line (e.g., infected by a virus, bacterial, mycoplasma, chlamydia) , a genetically defective cell or cell line. These target cells may be cell lines obtained from cell line banks. Alternatively, the cells can be obtained from an individual having a disease or a disorder. For example, target cells can be obtained from a tumor biopsy of a cancer patient.

Recombinant membrane proteins derived from cancer cells may include, but are not limited to, those from cells associated with Hodgkin's Disease, non-Hodgkin's B-cell lymphomas, T-cell lymphomas, malignant lymphoma, lymphosarcoma leukemia, chronic lymphocytic leukemia, multiple myeloma, chronic myeloid leukemia, chronic myelomonocytic leukemia, myelodysplastic syndromes, myeloproliferative disorders, hypereosinophilic syndrome, eosinophilic leukemia, multiple myeloma, X-linked lymphoproliferative disorders, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, pancreatic cancer and gallbladder cancer, cancer of the adrenal cortex, ACTH-producing tumor, bladder cancer, brain cancer (e.g., neuroblastomas and gliomas) , Ewing's sarcoma, head and neck cancer (e.g., mouth cancer and larynx cancer) , kidney cancer (e.g., renal cell carcinoma) , liver cancer, lung cancer (e.g., small and non-small cell lung cancers) , malignant peritoneal effusion, malignant pleural effusion, skin cancers (e.g., malignant melanoma, tumor progression of human skin keratinocytes, epithelial cell carcinoma, squamous cell carcinoma, basal cell carcinoma) , mesothelioma, Kaposi's sarcoma, bone cancer (e.g., osteomas and sarcomas such as fibrosarcoma and osteosarcoma) , cancers of the female reproductive tract (e.g., uterine cancer, endometrial cancer, ovarian cancer, and cervical cancer) , breast cancer, prostate cancer, retinoblastoma, testicular cancer, and thyroid cancer.

Recombinant membrane proteins derived from virally-infected cells can include but not limited to those derived from cells infected with coronavirus (such as SARS-CoV-2, such as RBD) , Epstein Barr Virus, HIV, influenza virus, polio virus, hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Varicella zoster virus, Rubella virus, measles virus, Herpes Simplex Virus, Dengue virus, papilloma virus, respiratory syncytial virus, or rabies virus.

Recombinant membrane proteins may derive from cells of patients with autoimmune diseases such as autoimmune thyroid disorders, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, immune haemolytic anaemia.

The effector cells used in the methods provided herein typically are cells that express one or more Fcγ receptors. In some embodiments, the FcγR is selected from FcγRI (e.g., FcγRIa, FcγRIb and FcγRIc) , FcγRII (e.g., FcγRIIa, FcγRIIb and FcγRIIc) and FcγRIII (e.g., FcγRIIIa or FcγRIIIb) , preferably FcγRII (such as FcγRIIa) . Preferably, the FcγR is FcγRIIIa.

Suitable effector cells include, but are not limited to, peripheral blood mononuclear cells (PBMCs) , natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils. In some embodiments, the effector cells used in the methods described herein are PBMCs. PBMCs are a mixture of monocytes and lymphocytes that can be isolated from whole blood using, for example, standard experimental protocols described in the art.

In some embodiments, the effector cells may comprise an antibody or a functional antibody fragment of thereof attached or conjugated to the surface of the cells.

In some embodiments, the effector cells further comprise one or more report genes selected from the group consisting of fluorescin (such as luciferase, GFP) , β-galactosidase, secreted alkaline phosphatase (SEAP) .

Method of the invention

Provided herein is an in vitro method for assessing and/or determining immune response induced by a biologic molecule.

In some embodiments, the method comprises:

b) contacting the immobilized protein or fragment with (i) a biologic molecule; and (ii) an effector cell, wherein the biologic molecule is capable or suspected to be capable of binding to the immobilized protein or fragment and the effector cell, and activating the effector cell;

In some embodiments, the support for the recombinant membrane protein or the functional fragment thereof is one or more selected from a plate (preferably multiwall plate for high throughput method) , a substrate, a column, a bead, a filter, a membrane, a tube and a chip. In some embodiments, the support is a Meso Scale Discovery (MSD) plate.

In some embodiments, the recombinant membrane protein has already been immobilized on the support, or is to be immobilized on the support before use (such as immediately before the assessment) .

In some embodiments, the contact of the biological molecule and the immobilized recombinant membrane protein and effector cell is carried out by adding the three biological components (such as sequentially or simultaneously) under a condition suitable for the interaction (such as binding) between them and incubating the same for a period sufficient for the interaction. For example, effector cells can be added to the medium before antibody, or antibody can be added before effector cells. The methods described herein can employ any combination of recombinant membrane protein, effector cells, and biological molecule, and the selections of those biological components used in the methods can depend on the purpose of the assay.

In some embodiments, the activation of the effector cell after contacting with the immobilized recombinant membrane protein and the biological molecule is detected. The detection can be carried out by detecting the changes in the expression levels of report genes, labels and/or biomarkers of the effector cells. In some embodiments, the report genes detected comprise but not limited to fluorescin (such as luciferase, GFP) , β-galactosidase, secreted alkaline phosphatase (SEAP) reporter gene) in the effector cell. In some embodiments, the detection to the labels may comprise but not limited to fluorescein labeling assay (such as dissociation-enhanced lanthanide fluorescence immunoassay assay (DELFIA) ; Calcein AM labeling) , enzyme labeling assay (such as lactose dehydrogenase assay) , radioactive labeling assay (such as Cr⁵¹) . In some embodiments, the biomarkers may comprise but not limited to CXCL9, CXCL10 and CXCL11 and UBD, IDO1, STEAP4, JAG1, APOL4, GBP4, CD274, GBP5, CCL3 and CCL4.

In some embodiments, the up-regulation in the expression level of the report gene, labels and/or biomarkers, compared to a reference expression level, is indicative of the ability of the antibody or the functional antibody fragment to affect ADCC function against the target cells.

In some other embodiments, a down-regulation or no change in expression level of the report gene, labels and/or biomarkers, compared to a reference expression level, is indicative of the inability of the antibody or the functional antibody fragment thereof to affect ADCC function against the target cells.

The reference level can be for example expression level of the report gene, labels and/or biomarkers determined before step b) or before co-incubation; determined with an irrelevant antibody; or a standard level previous determined.

In some embodiments, the method can be carried out in a high-throughput way. For example, the method may be used in simultaneously assessing the ability of multiple combinations of antibodies, effector cells, and recombinant proteins.

Use of the method and corresponding products

The method of the present disclosure and the corresponding products (such as a system or a kit for determining the immune effect) are useful in laboratory, manufacture and clinic. For example, the bioassays invented can be carried out in the analytical labs in the biopharmaceutical company for product quality assessment during product development and manufacturing. They can also be carried out in the clinical labs to measure the clinical responses in a patient.

In some embodiments, the method of the present disclosure can be used in screening a candidate biological molecule (such as an antibody or antibody-binding fragment thereof or a conjugate comprising the antibody or the fragment) based on the ability to induce immune response (such as ADCC and/or ADCP) against the membrane protein and in turn a target cell comprising the membrane protein.

In some embodiments, the method of the present disclosure can be used in controlling the quality of the biological molecule or product comprising the same (for example, an antibody or the functional antibody fragment thereof or a formulation comprising the same) , such as during the manufacture, storage, transportation and/or application of the biological molecule. For example, an abnormal expression level of the aforementioned report gene, labels and/or biomarkers, compared to a reference expression level (such as level in a quality guarantee range) , led by the antibody or antibody-binding fragment thereof is indicative of an unqualified antibody or the functional antibody fragment, and vise-versa.

In some embodiments, the method of the present disclosure can be used in screening a candidate compound for the ability to modulate immune response induced by the biological molecule (such as ADCC and/or ADCP) , wherein the method further comprises adding the candidate compound into the system of step a) or b) . For example, the up-regulation in the expression level of the report gene, labels and/or biomarkers, compared to the expression level determined without adding the candidate compound, is indicative of the ability of the candidate compound to up-modulate the ADCC function, and vise-versa.

In some embodiments, the method of the present disclosure can be used in optimizing the types (including combinations) and/or concentration of the biological molecule (e.g., the antibody or the functional antibody fragment thereof) in inducing immune response (such as ADCC and/or ADCP) to the membrane protein, and in turn the target cell expressing the membrane protein. For example, various types (including combinations) and/or concentration of antibody or the functional antibody fragment thereof can be added, and the optimization can be made based on the expression levels of the report gene, labels and/or biomarkers.

In some embodiments, the method of the present disclosure can be used in predicting the effect of the biological molecule (e.g., an antibody or the function antibody fragment thereof) in treatment of target membrane protein (and in turn target cell expressing the membrane protein) associated diseases. For example, the up-regulation in the expression level of the report gene, labels and/or biomarkers, compared to the expression level, is indicative of the ability of the antibody in inducing ADCC function, and vise-versa. If ADCC and/or ADCP is useful in treatment of the disease (such as cancer) , the antibody has treatment effect or improved treatment effect to the disease. If ADCC and/or ADCP is not benefit for the treatment of a disease (such as an autoimmune disease) , the antibody is not suggested to be used.

In some embodiments, the method of the present disclosure can be used in assessing the interactions between two or more antibodies and/or functional antibody fragments thereof in immune response (such as ADCC and/or ADCP) , wherein the two or more biological molecules are added to the same system.

It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

Also provided herein are products and systems relating to the present methods and uses.

In some embodiments, provided herein is a kit assessing and/or determining immune response induced by a biologic molecule, which comprises agents and/or devices for carrying out the method of the present method. In some embodiments, provided herein is a system for assessing and/or determining immune response induced by a biologic molecule, which comprises means for use in one or more steps of the method. In some embodiments, the system may be an automated operating system.

Various device components can be components as generally known in the art. For example, control electronics may be provided in any suitable form and may, for example, include memory and a processor. Processor may one or more components that can include any one or more of a microprocessor, a controller, a digital signal processor (DSP) , an application specific integrated circuit (ASIC) , a field-programmable gate array (FPGA) , equivalent discrete or integrated logic circuitry, programmable logic circuitry, or the like, and the functions attributed to processor herein may be embodied as hardware, firmware, software or any combination thereof. Memory may store instructions that cause processor to provide the functionality ascribed to programmer herein, and information used by processor to provide the functionality ascribed to programmer herein. Memory may include any volatile, non-volatile, magnetic, optical, or electrical media, such as a random access memory (RAM) , read-only memory (ROM) , non-volatile RAM (NVRAM) , electrically-erasable programmable ROM (EEPROM) , flash memory, or any other digital media. Memory may also store information that controls the parameters in the method.

Such hardware, software, firmware may be implemented within the same device or within separate devices to support the various operations and functions described in this disclosure. In addition, any of the described units, modules or components may be implemented together or separately as discrete but interoperable logic devices. Functionality associated with one or more modules or units may be performed by separate hardware or software components, or integrated within common or separate hardware or software components.

Exemplary materials and steps for carrying out the methods

The following are some Exemplary materials and steps for carrying out the methods. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

1. A method for assessing and/or determining antibody-dependent cell-mediated immune response comprising:

b) contacting the immobilized protein or fragment with (i) an antibody or an Fc-containing fragment thereof; and (ii) an effector cell;

2. The method of embodiment 1, wherein the antibody-dependent cell-mediated immune response is antibody-dependent cell-mediated cytotoxicity (ADCC) , Antibody-dependent trogocytosis (ADTC) and/or antibody-dependent cell-mediated phagocytosis (ADCP) .

3. The method of embodiment 1, wherein the recombinant membrane protein or the antibody binding fragment thereof is a disease-associated protein or fragment, and/or

wherein the recombinant membrane protein or the antibody binding fragment thereof is one or more expressed by a target cell (such as a tumor cell, an infected cell, a genetically defective cell and/or a pathogenicity immune cell) of the immune response;

for example, the recombinant membrane protein is one or more selected from CD20, HER2, RBD, CD38, and GD-2 protein.

4. The method of embodiment 1, wherein the support is a solid support; and/or

the support is one or more selected from a plate (such as multiwall plate) , a column, a bead, a filter, a membrane, a tube and a chip;

for example, the plate is a Meso Scale Discovery (MSD) plate.

5. The method of embodiment 1, wherein the antibody or the Fc-containing fragment thereof is capable of specifically binding to the recombinant membrane protein or the antibody binding fragment thereof; and/or

the antibody is a monoclonal antibody, a polyclonal antibody, a natural antibody, a genetically engineered antibody, for example, an antibody with genetically engineered Fc; and/or

the antibody is a chimeric antibody, a multivalent antibody, a humanized antibody, or a human antibody.

6. The method of embodiment 1, wherein the effector cell is an FcR expressing or over expressing effector cell;

for example, the FcR is a FcγR, for example FcγR selected from FcγRI (e.g., FcγRIa, FcγRIb and FcγRIc) , FcγRII (e.g., FcγRIIa, FcγRIIb and FcγRIIc) and FcγRIII (e.g., FcγRIIIa or FcγRIIIb) , preferably FcγRII (such as FcγRIIa) and FcγRIII (such as FcγRIIIa) ; and/or

the effector cell is selected from a natural killer (NK) cell, a peripheral blood mononuclear cell (PBMC) , a macrophagocyte, a cytotoxic T-lymphocyte, a neutrophil cell, and a dendritic cell; or an engineered cell, such as Jurkat cell, CHO cell, HEK 293 T cell expressing or overexpressing FcR.

7. The method of embodiment 1, wherein the activation of the effector cell is detected by the expression of a reporter gene (such as fluorescin (such as luciferase, GFP) , β-galactosidase, secreted alkaline phosphatase (SEAP) reporter gene) in the effector cell; and/or

the activation of the effector cell is detected by fluorescein labeling assay (such as dissociation-enhanced lanthanide fluorescence immunoassay assay (DELFIA) ; Calcein AM labeling) , enzyme labeling assay (such as lactose dehydrogenase assay) , radioactive labeling assay (such as Cr⁵¹) .

8. The method of embodiment 1, wherein the support is a MSD plate, the effector cell is a Jurkat/FcγR/NFAT-Luc cell, and the activation of the effector cell is detected by measuring luciferase activity represented by the luminescence.

9. The method of embodiment 1, wherein the method is used for:

(a') screening a candidate antibody or antigen-binding fragment thereof based on the ability to induce antibody-dependent cell-mediated immune response against a target cell expressing the membrane protein or an antibody binding fragment thereof;

(b') controlling the quality of the antibody or the fragment thereof, such as during the manufacture, storage, transportation and/or application of the antibody or fragment thereof;

(c') screening a candidate compound for the ability to modulate antibody-dependent cell-mediated immune response, wherein the method further comprises adding the candidate compound into the system of step b) ;

(d') optimizing the types and/or concentration of the antibody or the fragment thereof in inducing antibody-dependent cell-mediated immune response;

(e') predicting the effect of the antibody or the fragment thereof in treatment of target cell associated diseases; and/or

(f') assessing the interactions between two or more antibodies and/or functional antibody fragments thereof in antibody-dependent cell-mediated immune response, wherein the two or more antibodies and/or fragments thereof are used in step b) ; and/or

(g') evaluating the immune regulation ability of the membrane proteins or the fragment thereof to the antibody-dependent cell-mediated immune response induced by the antibody.

10. A product for assessing and/or determining antibody-dependent cell-mediated immune response comprising one or more substances selected from:

a") a support, a recombinant membrane protein or an antibody binding fragment thereof, substance for immobilizing the recombinant membrane protein or an antibody binding fragment thereof on a support; or, a support immobilized with a recombinant membrane protein or an antibody binding fragment thereof;

b") an antibody or an Fc-containing fragment thereof;

c") an effector cell;

d") optionally, substance for detecting the activation of the effector cell before, during and/or after contacting a support immobilized with a recombinant membrane protein or an antibody binding fragment thereof with b") and (c") .

11. The product of embodiment 10, wherein the product is a system (such as an automated operating system) or kit for assessing and/or determining antibody-dependent cell-mediated immune response, wherein the system or kit comprises means for carrying out the method of any of embodiments 1-9.

EXAMPLES

The following non-limiting examples are illustrative for the disclosure and are not to be construed as to be in any way limiting for the scope of the invention.

Publications cited herein and the materials for which they are cited are hereby specifically incorporated by reference in their entireties. All reagents, unless otherwise indicated, were obtained commercially. All parts and percentages are by weight unless stated otherwise. An average of results is presented unless otherwise stated. The abbreviations used herein are conventional, unless otherwise defined.

Materials and Methods

I. Bio-Materials

II. Methods

1. Immobilized recombinant proteins for eliciting ADCC

Solutions of recombinant CD20, HER2, or RBD proteins in PBS (pH7.4) at concentrations ranged from 2 μg/mL to 8 μg/mL were used to coat the wells of MULTI-SPOT 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) (100μL/well) . The plate was incubated overnight at 4 ℃ before removing the protein solutions, and then washed with PBS. The wells were blocked with PBS with 10%HI-FBS (Thermo Fisher Scientific, Shanghai, China) for 1-2 hours at 37℃.

The effector cell line was FcγRIIIa-over expressing Jurkat reporter cell line transfected with a firefly luciferase gene under the control of NFAT response elements. 4x10⁴ of effector cells were added to the wells and co-incubated with the immobilized recombinant proteins in the absence or presence of 0-10 μg/mL of Anti-CD20, Anti-HER2, or Anti-RBD therapeutic antibodies in RPMI1640 Media (Thermo Fisher Scientific, Shanghai, China) with 4%low lgG FBS (Thermo Fisher Scientific, Shanghai, China) for 6 hr at 37℃ in 5%CO₂.

The luciferase activities of the Jurkat/FcγRIIIa/NFAT-Luc cells were measured by using Bio-Glo Luciferase Assay System (Promega, Madison, WI) and the Envision Multimode Plate Reader (PerkinElmer, Waltham, MA) . The luminescence data representing effector cell activation were exported to the SoftMax Pro software (Molecular Devices, Sunnyvale, CA) and plotted against the Anti-CD20, Anti-HER2, or Anti-RBD antibody concentrations for a four-parameter analysis to estimate the ADCC activities of the antibodies.

2. Immobilized recombinant proteins for eliciting ADCP

Solution of recombinant CD20, HER2 (ARCO) , or RBD proteins in PBS (pH7.4) at a concentration of 1 μg/mL were used to coat the wells of MULTI-SPOT 96-well MSD plate (Meso Scale Discovery, Gaithersburg, MD) (100μL/well) . The plate was incubated overnight at 4 ℃ before removing the protein solutions, and then washed with PBS. The wells were blocked with PBS with 10%HI-FBS for 1-2 hours at 37 ℃.

The effector cell line was FcγRIIa-over expressing Jurkat reporter cell line transfected with a firefly luciferase gene under the control of NFAT response elements (GenScript Biotech, Nanjing, China) . 4x10⁴ of effector cells were added to the wells and co-incubated with the immobilized recombinant proteins in the absence or presence of 0-10 μg/mL of Anti-CD20, Anti-HER2, or Anti-RBD therapeutic antibodies in RPMI1640 Media with 4%low lgG FBS for 6 hr at 37℃ in 5%CO₂.

The luciferase activities of the Jurkat/FcγRIIa/NFAT-Luc cells were measured by using Bio-Glo Luciferase Assay System (Promega, Madison, WI) and the Envision Multimode Plate Reader (PerkinElmer) . The luminescence data representing effector cell activation were exported to the SoftMax Pro software (Molecular Devices, Sunnyvale, CA) and plotted against the Anti-CD20, Anti-HER2, or Anti-RBD antibody concentrations for a four-parameter analysis to estimate the ADCC activities of the antibodies.

3. Conventional Two-Cell Line ADCC Assay

CD20-expressing Raji cells (ATCC, Manassas, VA) , HER2-expressing BT474 cells (ATCC, Manassas, VA) , or S protein-expressing HEK293 cells (WuXi Biologics, Shanghai, China) were seeded at 2x10⁴-4x10⁴ per well in 96-well tissue culture plate before co-incubation with 4x10⁴ of aforementioned Jurkat/FcγRIIIa/NFAT-Luc cells per well in the absence or presence of anti-CD20, anti-HER2, or anti-RBD antibodies at 0-10 μg/mL in RPMI1640 Media with 4%low IgG FBS for 6 hr at 37℃ in 5%CO₂.

The luciferase activities of the Jurkat/FcγRIIIa/NFAT-Luc cells were measured by using Bio-Glo Luciferase Assay System (Promega, Madison, WI) and the Envision Multimode Plate Reader (PerkinElmer) . The luminescence data representing effector cell activation were exported to the SoftMax Pro software (Molecular Devices, Sunnyvale, CA) and plotted against the Anti-CD20, Anti-HER2, or Anti-RBD antibody concentrations for a four-parameter analysis to estimate the ADCC activities of the antibodies.

4. Conventional Two-Cell Line ADCP Assay

CD20-expressing Raji cells, HER2-expressing BT474 cells, or S protein-expressing HEK293 cells were seeded at 2x10⁴-4x10⁴ per well in 96-well tissue culture plate before co-incubation with 4x10⁴ of aforementioned Jurkat/FcγRIIa/NFAT-Luc cells per well in the absence or presence of anti-CD20, anti-HER2, or anti-RBD antibodies at 0-10 μg/mL in RPMI1640 Media with 4%low IgG FBS for 6 hr at 37℃ in 5%CO₂.

The luciferase activities of the Jurkat/FcγRIIa/NFAT-Luc cells were measured by using Bio-Glo Luciferase Assay System and the Envision Multimode Plate Reader (PerkinElmer) . The luminescence data representing effector cell activation were exported to the SoftMax Pro software (Molecular Devices, Sunnyvale, CA) and plotted against the Anti-CD20, Anti-HER2, or Anti-RBD antibody concentrations for a four-parameter analysis to estimate the ADCC activities of the antibodies.

Example 1. Comparison of ADCC elicited by immobilized recombinant proteins vs. target cells

ADCC effects elicited by immobilized recombinant proteins or target cell line were assessed using the above mentioned method and compared.

In the absence of CD20, there was no luminescence signal detected. In the presence of CD20 immobilized at 2μg/mL and as shown in Figure 1, in the presence of anti-CD20 antibody (Rituxan) , there was an antibody dose-dependent increase of Jurkat/FcγRIIIa/NFAT-Luc cell activation, represented by luminescence signals. Antibody dose-dependent ADCC response curves were also observed with immobilized HER2 (2 μg/mL) and RBD (8 μg/mL) in the presence of anti-HER2 antibody (Herceptin) and anti-RBD antibody (COVID NAb) , respectively.

Figure 2 shows the conventional ADCC assay results using both target and effector cell lines. CD20-, HER2-, and RBD-expressing target cells elicited similar antibody-dependent Jurkat/FcγRIIIa/NFAT-Luc cell activation compared to the one elicited by immobilized recombinant proteins and the same effector cells. The results indicate that immobilized recombinant proteins can simulate membrane proteins on the target cells to stimulate effector cells for an ADCC effect.

We also tried to immobilize the same recombinant proteins onto ELISA plate or common tissue culture plate, and found the MSD plate showed the best detection effect.

Example 2. Immobilized recombinant proteins allow accurate adjustment of immune stimulation

The relationship between the immobilized recombinant protein and immune stimulation of ADCC was further studied.

In the absence of recombinant protein, there was no ADCC effect observed. As shown in Figure 3, in the presence of approx. 0.25-8 μg/mL of immobilized CD20, HER2, or RBD antigens, 1 μg/mL of antibodies can effectively induce observable ADCC effect in an antigen dose-dependent manner.

This finding indicates that this immobilized recombinant protein model allows accurate adjustment of immune stimulation otherwise unlikely using the target cells with significantly less quantifiable antigen expression.

Example 3. Comparison of ADCP elicited by immobilized recombinant proteins vs. target cells

ADCP effects elicited by immobilized recombinant proteins or target cell line were assessed using the above mentioned method.

In the absence of CD20, there was no luminescence signal detected. As shown in Figure 4, in the presence of CD20 immobilized at 2 μg/mL and in the presence of anti-CD20 antibody (Rituxan) , there was an antibody dose-dependent increase of Jurkat/FcγRIIa/NFAT-Luc cell activation, represented by luminescence signals. Antibody dose-dependent ADCP response curves were much weaker with immobilized HER2 or RBD and in the presence of anti-HER2 antibody (Herceptin) and anti-RBD antibody.

Figure 5 shows the conventional ADCP assay results using both target cell line and effector cell line. CD20-expressing target cells elicited similar antibody-dependent Jurkat/FcγRIIa/NFAT-Luc cell activation compared to the one elicited by immobilized recombinant CD20 and the same effector cells. And the ADCP against HER2 or RBD was also much weaker, consistent with the results obtained using immobilized HER2 or RBD proteins. The above results indicate that immobilized recombinant proteins can simulate membrane proteins on the target cells to stimulate effector cells for an ADCP effect.

Example 4. Immobilized recombinant proteins allow accurate adjustment of immune stimulation

The relationship between the immobilized recombinant protein and ADCP immune stimulation was further studied.

In the absence of recombinant protein, there was no ADCP effect observed. As shown in Figure 6, in the presence of about 1-8 μg/mL of immobilized CD20 antigens, 1 μg/mL of antibodies can effectively induce observable ADCP effect in an antigen dose-dependent manner.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the compositions and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

REFERENCES:

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2. Hui E. Understanding T cell signaling using membrane reconstitution. Immunol Rev. 2019 Sep; 291 (1) : 44-56.

3. Bruhns P. Properties of mouse and human IgG receptors and their contribution to disease models. Blood. 2012; 119: 5640–5649.

4. Freeman SA, Goyette J, Furuya W, Woods EC, Bertozzi CR, Bergmeier W, Hinz B, van der Merwe PA, Das R, Grinstein S. Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis. Cell. 2016; 164: 128–140.

5. Boero S, Morabito A, Banelli B (2015) . Analysis of in vitro ADCC and clinical response to trastuzumab: possible relevance of FcγRIIIA/FcγRIIA gene polymorphisms and HER-2 expression levels on breast cancer cell lines. J Transl Med. 2015; 13: 324.

Claims

A method for assessing and/or determining immune response induced by a biologic molecule comprising:

a) immobilizing a recombinant membrane protein or a fragment comprising the extracellular domain thereof on a support;

b) contacting the immobilized protein or fragment with (i) a biologic molecule; and (ii) an effector cell, wherein the biologic molecule is capable or suspected to be capable of binding to the immobilized protein or fragment and the effector cell, and activating the effector cell;

c) detecting the activation of the effector cell so as to assess and/or determine the immune response induced by the biologic molecule.
The method of claim 1, wherein

the recombinant membrane protein is one or more proteins found in the target cell of the biologic molecule; and/or

the biologic molecule is capable of or is suspected to be capable of specifically binding to the recombinant membrane protein (such as is an antibody or a functional fragment thereof) ; and/or

the effector cell is a cell capable of being activated after binding to the biologic molecule; and/or

the support is a solid support.
The method of claim 2, wherein the recombinant membrane protein is a disease associated protein, and the biologic molecule is a therapeutic biologics; and/or

the recombinant membrane protein and the biologic molecule are a couple of binding partners, for example an antigen and an antibody, a receptor and a ligand, a substrate and an enzyme; and/or

the target cell is selected from a tumor cell, an infected cell, a genetically defective cell and/or a pathogenicity immune cell; and/or

the support is one or more selected from a plate (such as multiwall plate) , a substrate, a column, a bead, a filter, a membrane, a tube and a chip, for example, the plate is a Meso Scale Discovery (MSD) plate.
The method of claim 1, wherein the activation of the effector cell is detected by the expression of a reporter gene (such as fluorescin (such as luciferase, GFP) , β-galactosidase, secreted alkaline phosphatase (SEAP) reporter gene) in the effector cell; and/or

the activation of the effector cell is detected by fluorescein labeling assay (such as dissociation-enhanced lanthanide fluorescence immunoassay assay (DELFIA) ; Calcein AM labeling) , enzyme labeling assay (such as lactose dehydrogenase assay) , radioactive labeling assay (such as Cr⁵¹) .
The method of claim 1 for assessing and/or determining antibody-dependent cell-mediated immune response comprising:

a) immobilizing a recombinant membrane protein or an antibody binding fragment thereof on a support;

b) contacting the immobilized protein or fragment with (i) an antibody or an Fc-containing fragment thereof; and (ii) an effector cell;

c) detecting the activation of the effector cell so as to assess and/or determine the antibody-dependent cell-mediated immune response induced by the antibody or the fragment thereof.
The method of claim 5, wherein the antibody-dependent cell-mediated immune response is antibody-dependent cell-mediated cytotoxicity (ADCC) , Antibody-dependent trogocytosis (ADTC) , and/or antibody-dependent cell-mediated phagocytosis (ADCP) .
The method of claim 5, wherein the recombinant membrane protein or the antibody binding fragment thereof is a disease-associated protein or fragment, and/or

wherein the recombinant membrane protein or the antibody binding fragment thereof is one or more expressed by a target cell (such as a tumor cell, an infected cell, a genetically defective cell and/or a pathogenicity immune cell) of the immune response;

for example, the recombinant membrane protein is one or more selected from CD20, HER2, RBD, CD38, and GD-2 protein.
The method of claim 5, wherein the support is a solid support; and/or

the support is one or more selected from a plate (such as multiwall plate) , a column, a bead, a filter, a membrane, a tube and a chip;

for example, the plate is a Meso Scale Discovery (MSD) plate.
The method of claim 5, wherein the antibody or the Fc-containing fragment thereof is capable of specifically binding to the recombinant membrane protein or the antibody binding fragment thereof; and/or

the antibody is a monoclonal antibody, a polyclonal antibody, a natural antibody, a genetically engineered antibody, for example, an antibody with genetically engineered Fc; and/or

the antibody is a chimeric antibody, a multivalent antibody, a humanized antibody, or a human antibody.
The method of claim 5, wherein the effector cell is an FcR expressing or over expressing effector cell;

for example, the FcR is a FcγR, for example FcγR selected from FcγRI (e.g., FcγRIa, FcγRIb and FcγRIc) , FcγRII (e.g., FcγRIIa, FcγRIIb and FcγRIIc) and FcγRIII (e.g., FcγRIIIa or FcγRIIIb) , preferably FcγRII (such as FcγRIIa) and FcγRIII (such as FcγRIIIa) ; and/or

the effector cell is selected from a natural killer (NK) cell, a peripheral blood mononuclear cell (PBMC) , a macrophagocyte, a cytotoxic T-lymphocyte, a neutrophil cell, and a dendritic cell; or an engineered cell, such as Jurkat cell, CHO cell, HEK 293 T cell expressing or overexpressing FcR.
The method of claim 5, wherein the activation of the effector cell is detected by the expression of a reporter gene (such as fluorescin (such as luciferase, GFP) , β-galactosidase, secreted alkaline phosphatase (SEAP) reporter gene) in the effector cell; and/or

the activation of the effector cell is detected by fluorescein labeling assay (such as dissociation-enhanced lanthanide fluorescence immunoassay assay (DELFIA) ; Calcein AM labeling) , enzyme labeling assay (such as lactose dehydrogenase assay) , radioactive labeling assay (such as Cr⁵¹) .
The method of claim 5, wherein the support is a MSD plate, the effector cell is a Jurkat/FcγR/NFAT-Luc cell, and the activation of the effector cell is detected by measuring luciferase activity represented by the luminescence.
The method of claim 1 or 5, wherein the method is used for:

(a') screening a candidate biologic molecule based on the ability to induce immune response against a target cell expressing the membrane protein or an antibody binding fragment thereof;

(b') controlling the quality of the biologic molecule, during the manufacture, storage, transportation and/or application of the antibody or fragment thereof;

(c') screening a candidate compound for the ability to modulate immune response, wherein the method further comprises adding the candidate compound into the system of step b) ;

(d') optimizing the types and/or concentration of the biologic molecule in inducing antibody-dependent cell-mediated immune response;

(e') predicting the effect of the biologic molecule in treatment of target cell associated diseases; and/or

(f') assessing the interactions between two or more biologic molecules in immune response, wherein the two or more biologic molecule are used in step b) ; and/or

(g') evaluating the immune regulation ability of the membrane proteins or the fragment thereof to the immune response induced by the biologic molecule.
The method of claim 13, wherein the biologic molecule is an antibody or antigen-binding fragment thereof; and the immune response is an antibody-dependent cell-mediated immune response.
A product for assessing and/or determining immune response comprising one or more substances selected from:

a") a support, a recombinant membrane protein or a fragment comprising the extracellular domain thereof, substance for immobilizing the recombinant membrane protein or fragment thereof on a support; or, a support immobilized with a recombinant membrane protein or a fragment comprising the extracellular domain thereof;

b") a biological molecule capable or suspected to be capable of bind to the immobilized protein or fragment and activate an effector cell;

c") the effector cell;

d") optionally, substance for detecting the activation of the effector cell before, during and/or after contacting a support immobilized with a recombinant membrane protein or the fragment thereof with b") and c") .
The product of claim 15, wherein the biologic molecule is an antibody or antigen-binding fragment thereof; and the immune response is an antibody-dependent cell-mediated immune response.
The product of claim 15, wherein the product is a system (such as an automated operating system) or kit comprising means and/or substances for carrying out the method of any of claims 1-14.