Compositions and methods for determining t cell adapter effects

Abstract

An assay system for measuring the binding and dissociation of T cells mediated by T cell adapter (TCE) antibodies to target cancer cells, and its subsequent effects on T cell activation and proliferation. The assay platform can be used to screen TCE candidates that form detachable immune synapses and can provide long term protection.

Description

[0001] Cross-references to related applications This application claims the benefit of U.S. Provisional Application Serial No. 63 / 459,937, filed April 17, 2023, under 35 USC 119(e), the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] This application relates to methods for cancer detection. In particular, this application relates to T-cell engagers (TCEs). Background Technology

[0003] The cytotoxic nature of T cells makes them valuable in cancer treatment. Various strategies have been devised to utilize T cells in cancer therapy, such as T cell engagers (TCEs), chimeric antigen receptor (CAR) T cells, and TCR-T cells. TCEs, in particular, have proven highly potent: they induce cytotoxicity at picomolar doses and exhibit strong T cell activation. The first anti-CD3 / CD19 TCE, bonnetumab (Amgen, BLINCYTO®), was approved in 2014 for the treatment of acute lymphoblastic leukemia. Multiple studies have demonstrated that TCEs induce T cell activation, manifested as upregulation of CD25 and CD69 and the secretion of cytokines such as IL-2, TNF-α, and IFN-γ. Recently, a novel gp100 peptide-HLA TCR / anti-CD3 fusion protein, tembfos (Immunocore, KIMMTRAK®), was approved in 2022 for the treatment of metastatic uveal melanoma. The approval of TCEs underscores the need for more effective immunotherapies. However, although TCE is a potent cytotoxic agent, little is known about the long-term effects of TCE on T cells, and there is no evidence that it has a long-term protective effect against cancer recurrence. Summary of the Invention

[0004] The following description of the invention is illustrative only and is not intended to be limiting in any way. Other aspects, embodiments, and features will become apparent from the accompanying drawings and the following detailed description, in addition to the illustrative aspects, embodiments, and features described above.

[0005] This application provides, in particular, assay systems, apparatus, methods, and processes for evaluating the efficacy or safety of T-cell engagers (TCEs). This application also provides biological complexes and compositions.

[0006] In one aspect, this application provides an assay system for evaluating the efficacy and safety of T-cell engagers (TCEs). In one embodiment, the assay system includes a cell culture plate having at least one chamber for seeding multiple cells expressing at least one cancer biomarker in a cell culture medium. The density can be [per cm³ per chamber]. 2 At least 30,000, 40,000, 50,000, or 100,000 cells. These cells are attached to the surface of the cell culture plate.

[0007] The cell culture medium contains multiple T cells and a TCE. In one embodiment, the number of T cells may not exceed two-thirds (V) of the number of cancer cells inoculated. The concentration of TCE may be at least 1 ng per ml of cell culture medium. The TCE may have a first binding specificity for cancer markers and a second binding specificity for T cells.

[0008] In one implementation, the rate at which T cells attach to or detach from cells expressing at least one cancer marker can be measured.

[0009] In one embodiment, the assay system includes a first cell culture plate having at least one chamber for seeding a plurality of cells expressing at least one cancer biomarker in a first cell culture medium. The density can be per cm³ per chamber. 2 At least 30,000, 50,000, or 100,000 cells. These cells can attach to the surface of the first cell culture plate.

[0010] In one embodiment, the first cell culture medium contains a plurality of T cells and a TCE. The number of T cells may be approximately 4, 5, or 6 times the number of inoculated cancer cells. The concentration of the TCE may be at least 1 ng per ml of the first cell culture medium. The TCE may have a first binding specificity for a cancer biomarker and a second binding specificity for T cells. Cells expressing at least one cancer biomarker, T cells, and TCE are co-cultured for a co-culture period to provide activated T cells.

[0011] In one embodiment, the TCE can be configured to bind T cells and cells expressing at least one cancer biomarker to provide a biological complex that activates the T cells, kills the cells expressing at least one cancer biomarker, and detaches the cells expressing at least one cancer biomarker from a first cell culture plate. In one embodiment, the biological complex containing T cells may initially attach to the first cell culture plate due to the attachment of the cells expressing at least one cancer biomarker. The activated T cells are then detached from the first culture plate and released into a first cell culture medium by killing the cells expressing at least one cancer biomarker. Thus, in one embodiment, the first cell culture medium may contain activated T cells detached from the first cell culture plate by killing the cells expressing at least one cancer biomarker.

[0012] The assay system also includes a second cell culture plate for measuring the growth rate of activated T cells. In one embodiment, the second cell culture plate contains a second cell culture medium. In one embodiment, activated T cells are removed from a first cell culture medium and cultured in the second cell culture medium for an expansion period to provide expanded activated T cells. In one embodiment, the growth rate is measured by pre-labeling T cells with a fluorescent dye and counting the number of expanded activated T cells by measuring, for example, fluorescence.

[0013] The assay system may further include a third cell culture plate for measuring the cytotoxicity of activated T cells. In one embodiment, the third cell culture plate contains cells expressing at least one cancer biomarker in a third cell culture medium. In one embodiment, activated T cells are removed from a first or second cell culture medium and co-grown with cells expressing at least one cancer biomarker in a third cell culture medium for a co-growth phase. In one embodiment, the cytotoxicity of activated T cells is measured by counting cell deaths in cells expressing at least one cancer biomarker.

[0014] In one implementation, the co-cultivation period is approximately half a day to approximately two days.

[0015] In one implementation, the amplification culture period is approximately 2 to approximately 7 days.

[0016] In one implementation, the symbiotic period is approximately half a day to approximately one day.

[0017] In one embodiment, the cells expressing the cancer biomarker include cancer cells. In one embodiment, the cancer biomarker includes CDH17. In one embodiment, the cancer cells include gastrointestinal cancer cells, gastric cancer cells, colorectal cancer cells, esophageal cancer cells, pancreatic cancer cells, hepatobiliary cancer cells, small cell lung cancer cells, ovarian cancer cells, bladder cancer cells, neuroendocrine tumor cells, adenomatous cancer cells, or combinations thereof.

[0018] In one embodiment, T cells include T lymphocytes derived from peripheral blood mononuclear cells, cytotoxic T cells, helper T cells, natural killer T cells, or combinations thereof. In one embodiment, T cells express CD3.

[0019] In one embodiment, the TCE includes a bispecific antibody or a multispecific antibody. In one embodiment, the bispecific antibody has binding specificity for both CD3 and CDH17.

[0020] In another embodiment, this application provides a biological complex. In one embodiment, the biological complex essentially comprises an immune synapse. In one embodiment, the biological complex comprises a TCE that binds to T cells and cells expressing at least one cancer biomarker. In one embodiment, the biological complex comprises a TCE that binds to T cells. In one embodiment, the biological complex comprises a TCE that binds to cells expressing at least one cancer biomarker.

[0021] In one embodiment, the biological complex comprises a TCE that binds to T cells and cancer cells, wherein the cancer cells are gastrointestinal (GI) cancer cells.

[0022] In another aspect, this application provides a method for evaluating the efficacy of a T-cell binder (TCE). In one embodiment, this application provides the steps of: attaching a plurality of cells expressing at least one cancer biomarker to a cell culture plate, introducing a plurality of T cells into a cell culture medium, and introducing a TCE into the cell culture medium. In one embodiment, the TCE has a first binding specificity for the cancer biomarker and a second binding specificity for T cells, thus the TCE is configured to bind to T cells and cells expressing at least one cancer biomarker.

[0023] The method also includes measuring the rate at which T cells attach to or detach from cells expressing at least one cancer marker, or measuring the rate at which T cells attach to or detach from a cell culture plate.

[0024] In another embodiment, this application provides the following steps: attaching a plurality of cells expressing at least one cancer biomarker to a first cell culture plate and culturing the cells in a first cell culture medium for a cell culture period; introducing a plurality of T cells into the first cell culture medium; and introducing a TCE into the first cell culture medium. In one embodiment, the TCE has a first binding specificity for the cancer biomarker and a second binding specificity for T cells, and therefore the TCE is configured to bind to T cells and cells expressing at least one cancer biomarker.

[0025] The method further includes the step of co-culturing T cells that bind to cells expressing at least one cancer marker attached to a first cell culture plate via TCE. In this state, T cells can attach to the first cell culture plate via plate-binding cells expressing at least one cancer marker.

[0026] In one embodiment, the method further includes the steps of: collecting activated T cells detached from a first cell culture plate (and thus suspended in the supernatant of a first cell culture medium), and allowing the activated T cells to grow for an expansion culture period in a second cell culture plate containing a second cell culture medium to provide expanded activated T cells, pre-labeling the T cells with a fluorescent dye, and counting the number of expanded activated T cells to provide the growth rate of the T cells.

[0027] In one embodiment, the method further includes the steps of: growing activated T cells in a co-occurrence phase in a third cell culture plate containing cells expressing at least one cancer biomarker; and measuring the cytotoxicity of the activated T cells by counting cell deaths of the cells expressing at least one cancer biomarker.

[0028] In one implementation, TCE is a bispecific antibody. In one implementation, the cells expressing the cancer marker are cancer cells. Attached Figure Description

[0029] The foregoing and other features of this disclosure will become more apparent from the accompanying drawings, the following description, and the appended claims. It should be understood that these drawings depict only a few embodiments arranged according to this disclosure and are therefore not intended to limit its scope. This disclosure will be described with further specificity and detail using the drawings, in which: Figure 1 A schematic diagram illustrates a assay for measuring the attachment / detachment rate of T cells to cancer cells driven by TCE. (A) T cells are added to adherent cancer cells, and cell counts are performed in the culture medium collected from the wells at different time points. The number of unbound T cells in the culture medium decreases over time. This assay measures the T cell attachment rate. (B) After T cells attach to cancer cells, unbound T cells are washed away, leaving bound cells. Fresh culture medium containing TCE is then added, and the bound T cells are allowed to detach slowly. Cell counts are performed in the culture medium collected from the wells at different time points. The number of unbound T cells in the culture medium increases over time. This assay measures the T cell detachment rate.

[0030] Figure 2 depicts the binding and dissociation curves of T cells to cancer cells. (A) Cell binding curves at various ARB202 concentrations. Higher antibody concentrations result in faster binding and slower dissociation. (B) Half-life calculated from the curves, and the same trend is observed. (C) Cell binding curves of ARB203 and the lower affinity D124A point mutant. The D124A mutant has a similar binding rate but faster dissociation. (D) Half-life calculated from the curves, the dissociation rate of the D124A mutant is twice that of ARB203; Figure 3 depicts the affinity, cell binding kinetics, and functionality of the TCE variants. (A) ELISA affinity of the anti-CD3 variant and adaptor variant of ARB202 with CDH17 and CD3 antigens. (B) Schematic diagram of the adaptor variant. (C) Cell binding curves of the anti-CD3 variant. It was observed that higher CD3 affinity resulted in faster binding and slower dissociation. (D) Half-life calculated from the curves, and the same trend was observed. (E) Cytotoxicity and IFN-γ production of the variants. Higher CD3 affinity led to strong cytotoxicity and T cell activation, while the adaptor had no effect on functionality.

[0031] Figure 4 illustrates the repeated killing assay. (A) T cells were cultured with cancer cells in the presence of TCE for 3 days to kill them, then collected and cultured for another 5 days in the absence of cancer cells. These activated T cells were then subjected to a second round of killing in the absence of TCE, and cytotoxicity was measured using a calcein-AM assay. (B) The efficacy of the second round of killing was correlated with the TCE concentration of the first round; and Figure 5 illustrates T cell proliferation after cytotoxicity. (A) T cells were labeled with a cell-tracing dye (e.g., CFSE) and cultured with cancer cells for 2 days for cytotoxicity, then collected and cultured for another 5 days in cancer-free conditions. T cell viability and FITC fluorescence were then analyzed by flow cytometry. (B) Significantly higher T cell counts and lower FITC MFI were observed in the ARB202-treated group, indicating T cell proliferation following cancer cell activation. (C) A dose-dependent increase in CFSE-low expressing (divided) T cells and Ki67+ T cells was observed in proliferation following ARB203 cytotoxicity. Detailed Implementation

[0032] In the following detailed description, reference is made to the accompanying drawings, which form a part of the description. In the drawings, similar symbols generally identify similar components unless the context otherwise requires. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It is readily understood that aspects of this disclosure (as shown in the general description and drawings herein) can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are expressly contemplated herein.

[0033] This application generally relates, and particularly to compositions, methods, apparatuses, systems, devices, and / or computer program products relating to the characterization of multiple T cells.

[0034] In T-cell therapy, multiple cytotoxic T cells can initially be derived from peripheral blood mononuclear cells (PBMCs), which are regulated by T-cell receptor complex proteins and co-stimulatory signaling proteins via agonist or antagonist receptors on their surface. Despite the promising prospects of TCE antibodies in immunotherapy for cancer treatment, many challenges remain. For example, TCE antibodies may bind to normal tissues expressing low levels of tumor-associated antigens (TAAs), leading to toxicity, i.e., their potential on-target detumescent toxicity. Another well-known issue relates to the control of T-cell activation. Overactivation of T cells can lead to systemic inflammation, such as potentially life-threatening cytokine release syndromes. Treating solid tumors is often challenging due to tumor heterogeneity and the tumor microenvironment. Each engineered TCE requires validation through a variety of in vitro, ex vivo, and in vivo experiments to demonstrate that each TCE antibody can penetrate the matrix, contact tumor cells, and induce T-cell toxicity against them.

[0035] In addition to the challenges mentioned above, research on TCEs has primarily focused on short-term killing via T cell attachment, with the ability of T cells to detach from target cells and undergo clonal activation remaining unclear. If T cells remain attached to target cells during killing, they may undergo apoptosis along with the target; if T cells detach, they can bind to another target and potentially expand clonally. Therefore, the formation of detachable flexible immune synapses is crucial for the short-term and long-term efficacy of TCEs. To assess whether TCE candidates can form detachable immune synapses, this application discloses a composition of an assay platform and a quantifiable method for biopharmaceutical companies to measure the binding kinetics of T cells to target cells. Specifically, the disclosed method measures the long-term effects of TCE stimulation. Among several assay parameters, the measurement of effector cell detachment allows for the clonal expansion of memory T cells targeting tumor cells. Evidence of T cell memory formation reveals a novel mechanism of action (MoA) that may have practical implications for TCE therapeutics. In this context, the formation of T cell memory targeting tumor antigens not only improves immune surveillance but also reduces the risk of post-treatment relapse.

[0036] As used herein, the terms “a”, “an”, and “the” are defined as meaning “one or more” and include the plural form unless the context is inappropriate.

[0037] As used herein, the terms “peptide” and “protein” are used interchangeably and are defined as referring to a biomolecule composed of amino acids linked together by peptide bonds.

[0038] The term "antigen" refers to an entity or segment thereof that can induce an immune response in an organism, particularly animals, and more particularly mammals, including humans. This term includes immunogens and the regions or antigenic determinants responsible for antigenicity.

[0039] The term "antibody" is used in the broadest sense to specifically include monoclonal antibodies and / or recombinant antibodies (including agonist and antagonist antibodies), antibody compositions with multi-epitope specificity, and antibody fragments (e.g., Fab, F(ab')2, and Fv), provided they exhibit the desired biological activity. In some embodiments, antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-chain antibodies, multispecific or pleiotropic antibodies, human antibodies, and humanized antibodies, as well as their active fragments. Examples of active fragments of molecules that bind to known antigens include Fab, F(ab')2, scFv, and Fv fragments, including products of Fab immunoglobulin expression libraries and epitope-binding fragments of any of the antibodies and fragments described above.

[0040] The term "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of a tightly bound, non-covalently bound heavy-chain variable domain and a light-chain variable domain. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. The six CDRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, although with lower affinity than the entire binding site.

[0041] In some implementations, antibodies may include immunoglobulin molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing binding sites that specifically bind to antigens. A typical antibody is a heterotetrameric protein that typically comprises two heavy (H) chains and two light (L) chains. Each heavy chain consists of a heavy chain variable domain (abbreviated as VH) and a heavy chain constant domain. Each light chain consists of a light chain variable domain (abbreviated as VL) and a light chain constant domain. Based on the amino acid sequence of the constant domain, the light chains of antibodies (immunoglobulins) from any vertebrate species can be classified into one of two distinct types, called κ and λ. The VH and VL regions can be further subdivided into highly variable complementarity-determining region (CDR) domains and more conserved regions called framework regions (FRs). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Binding regions that interact with antigens exist within the variable regions of both the light and heavy chains.

[0042] Immunoglobulins can be classified into different classes based on the amino acid sequence of their heavy chain constant domains. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; and IgA-1 and IgA-2. The heavy chain constant domains corresponding to different classes of immunoglobulins are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well-known.

[0043] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for a possible small number of naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, each monoclonal antibody targets a single determinant on the antigen, unlike conventional (polyclonal) antibody formulations which typically contain different antibodies targeting different determinants (epitopes). In addition to specificity, monoclonal antibodies have the advantage of being synthesized from hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody derived from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies used according to this disclosure can be prepared by the hybridoma method first described in Kohler & Milstein, Nature, 256:495 (1975), or by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Recombinant" means that the antibody is generated in a heterologous host cell using recombinant nucleic acid technology.

[0044] Monoclonal antibodies can be produced using a variety of methods, including but not limited to mouse hybridoma, phage display, recombinant DNA, molecular cloning of antibodies directly from primary B cells, and antibody discovery methods (see Siegel. Transfus. Clin. Biol. 2002; Tiller. New Biotechnol. 2011; Seeber et al., PLOS One. 2014). Monoclonal antibodies can include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity (US Patent No. 4,816,567; and Morrison et al.). Proc. Natl. Acad. Sci. USA, 81:6851-6855

[1984] ).

[0045] As used herein, a "multispecific" antibody means an antibody having at least two binding sites, each binding site having binding affinity for an epitope of an antigen. As used herein, a "bispecific, trispecific, tetraspecific, pentaspecific, or hexaspecific" antibody means an antibody having two, three, four, five, or six antigen binding sites. For example, the antibody disclosed herein with five binding sites is pentaspecific, and the antibody with six binding sites is hexaspecific.

[0046] As used herein, the terms “cancer biomarker” and “tumor-associated antigen (TAA)” are interchangeable and are defined as any substance present in or produced by cancer cells or other cells in the body that responds to cancer or certain benign (non-cancerous) conditions, providing information about cancer, such as how aggressive it is, what kind of treatment it may respond to, or whether it responds to treatment.

[0047] As used herein, the terms “immune synapse” and “immune synapse” are interchangeable and are defined as the interface between antigen-presenting cells or target cells and lymphocytes (such as T cells, B cells, or natural killer cells).

[0048] As used herein, the terms “T cell engager,” “T-cellengager,” and “TCE” are interchangeable and are defined as referring to therapeutic agents used in T-cell engagement therapy. TCEs are antibodies engineered to redirect the immune system’s T cells to recognize and kill cancer cells. They are designed to bind to target antigens expressed on cancer cells and triggering molecules on T cells, such as CD3. These therapeutic molecules then engage T cells present in the tumor but unable to recognize cancer cells, redirecting their activity to the tumor.

[0049] Bispecific T-cell binders (BiTEs) are a class of artificial bispecific monoclonal antibodies investigated for potential use as anticancer drugs. They guide the host's immune system, more specifically, the cytotoxic activity of T cells, to fight cancer cells. BiTE is a registered trademark of Micromet AG (a wholly owned subsidiary of Amgen Inc). BiTEs are fusion proteins composed of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, located on a single polypeptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, and the other binds to tumor cells via a tumor-specific molecule. Like other bispecific antibodies, and unlike ordinary monoclonal antibodies, BiTEs form a link between T cells and tumor cells. This allows T cells to exert cytotoxic activity against tumor cells by producing proteins such as interferon-γ, perforin, and granzymes, independent of the presence of MHC class I molecules or co-stimulatory molecules. These proteins enter tumor cells and initiate apoptosis.

[0050] The first BiTE, bonnetumab (Amgen, BLINCYTO®), has been approved by the FDA for the treatment of patients with relapsed and / or refractory B-cell precursor acute lymphoblastic leukemia (B-ALL). To expand the indications for BiTE therapy from hematopoietic malignancies to common solid tumors, new and more effective TCE therapies and rational combination approaches are under investigation, including bifunctional checkpoint inhibitory T-cell binders (CiTE), synchronizing multiple interacting T-cell binders (SMITE), trispecific killer binders (TriKE), and biTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells). The elucidation of resistance and immune escape mechanisms, along with new technologies used in drug development, paves the way for integrating various immune functions into a single molecule or single-cell carrier, thereby enhancing efficacy without compromising safety.

[0051] This disclosure can be more readily understood by referring to the following detailed description of the specific embodiments and examples included herein. Although this disclosure has been described with reference to specific details of certain embodiments, it is not intended to be construed as limiting the scope of this disclosure.

[0052] Example Example 1. Bispecific TCEs: ARB202, ARB203 and variants.

[0053] As a representative of TCEs, BiTE molecules are bispecific antibodies designed to form a bridge between cancer cells and cytotoxic T cells. A typical bispecific BiTE antibody has one domain that binds CD3 and another that binds a tumor-associated antigen (TAA). CD3 is a protein complex and T cell co-receptor involved in activating cytotoxic T cells (CD8+ naïve T cells) and helper T cells (CD4+ naïve T cells). To demonstrate how the attachment-detachment assay works, two sets of bispecific antibodies were used. The first set included ARB202—a CDH17 / CD3 bispecific antibody (see WO2019222428A1, which is incorporated herein by reference in its entirety), along with its CD3 variant and adaptor variant (see Table 1). The second set included ARB203—a Trop2 / CD3 bispecific antibody (see WO2021113748A1, which is incorporated herein by reference in its entirety), along with its low-affinity mutant (with D124A).

[0054] For the adapter variant, the adapter sequence was adapted from a published article (Chen, Zaro & Shen, 2013). To generate a lower affinity antibody variant, a point mutation was performed at the complementary site. The complementary site was derived using proABC-2, an algorithm for predicting amino acid residues in an antibody that participate in antibody-antigen interactions (Ambrosetti et al., 2020).

[0055] Antigen binding of the variant was characterized by ELISA. A fixed amount of antigen was coated in wells. The antigen and antibody were then incubated at a specific molar ratio. EC50 was then calculated based on the signal emitted by the binding antibody. To characterize the cytotoxicity of the variant, cancer cells expressing the target biomarker were co-cultured with T cells in the presence of a series of different antibody concentrations. Cancer cell viability was assessed at a specific endpoint by calcein-AM staining. The supernatant of the co-culture was also collected so that IFN-γ released by T cells during co-culture could be quantified using ELISA.

[0056] Example 2. Attachment / Detachment Dynamics Cancer cells were seeded into wells, and fluorescently labeled T cells and TCEs were added. The T cells bound to the cancer cells, thus reducing the number of unbound free T cells in the culture medium. At the desired time points, the culture medium was collected, and cell counting was performed by flow cytometry to calculate the fraction of attached T cells. Figure 1 A). For detachment kinetics, T cells were first allowed to attach for one hour, then unbound cells were removed and the medium was replaced with fresh medium containing TCE. Bound cells detached slowly into the new medium, and the medium was collected at time points for cell counting. Figure 1 B). The attachment / detachment half-life can be calculated from the generated curve. This assay demonstrates that the CDH17 / CD3 bispecific antibody ARB202 induces dose-dependent binding and dissociation of T cells from DLD1 cells. Figure 2A and 2B The Trop2 / CD3 bispecific antibody ARB203 and its low-affinity Trop2 point mutant D124A also showed a 2-fold difference in dissociation, but no difference in binding rate. Figure 2C and 2D On the other hand, for the ARB202 anti-CD3 variant with reduced affinity for CD3 (… Figure 3A ), and it was observed that the binding of T cells to CDH17+ cancer cells decreased while dissociation increased. Figure 3B and 3C This is consistent with the reduced cytotoxicity and decreased IFN-γ production observed in anti-CD3 variants. Figure 3E In addition to the anti-CD3 variant, a connector variant of ARB202 was also generated. Figure 3BThese adapter variants enable T cells to bind and dissociate at rates similar to wild-type ARB202. Figure 3A This is consistent with the fact that these adaptor variants exhibit similar cytotoxicity to wild-type ARB202 when co-cultured with CDH17+ cancer cell lines. Figure 3E Table 1 summarizes the characterization of ARB202 and its CD3 variants and adaptor variants for comparison, including CD3 / CDH17 binding EC50, binding half-life, dissociation half-life, cytotoxic EC50, and IFN-γ EC50, as measured by fold differences.

[0057] Example 3. Repeated cytotoxicity T cells and cancer cells were co-cultured with TCE for 24 hours to induce cell killing. T cells were then collected from the culture medium and cultured in RPMI medium containing IL-2 for up to one week. In a series of E:T cycles, activated T cells and cancer cells were co-cultured a second time for 24 hours without TCE. Figure 4A The amount of viable cancer cells was determined by calcein-AM staining. T cells activated with a higher concentration of ARB202 in the first round showed stronger cytotoxicity in the second round in the absence of ARB202. Figure 4B ).

[0058] Example 4. Proliferation of T cells after cytotoxicity T cells were stained with CFSE and co-cultured with cancer cells and TCE for 24 hours to induce cell killing. T cells were then collected from the culture medium and cultured in RPMI medium containing IL-2 for up to one week. T cells were collected, and the viability and CFSE signaling of the remaining viable cells were analyzed using flow cytometry. Figure 5A The dose-dependent increase in live T cell count and the decrease in mean fluorescence indicate that ARB202 activation leads to T cell expansion. Figure 5B The dose-dependent increase in CFSE-low expression T cells and Ki67+ T cells indicates that ARB203 has the ability to induce T cell proliferation and activation. Figure 5C ).

[0059] Example 5. Materials, Methods, and Schemes Adhesion-detachment measurement 1. In a 48-well plate, seed 30,000-120,000 cancer cells expressing the target biomarker into n wells, with a seeding density of 1 cm². 2 At least 30,000, and n = the number of time points involved in the attachment kinetics assessment.

[0060] 2. Stain T cells with a fluorescent dye. Add the stained T cells and 1-2000 ng / mL TCE to the wells, ensuring that the number of T cells does not exceed ⅔ of the number of cancer cells inoculated. Incubate the cells at 37°C and 5% CO2.

[0061] [Adhesion Dynamics Assessment] 3. Collect all supernatant from one well at the desired time points within the first hour after T cell addition. Wash the wells with 100–400 μL of PBS. Collect the PBS and combine it with the supernatant.

[0062] [Dissociation Dynamics Assessment] 4. At the final time point, fresh medium containing the same concentration of TCE was added back to the wells to assess attachment kinetics. Cells were incubated at 37°C and 5% CO2.

[0063] 5. Collect all supernatant at the desired time points within the following 3 hours. Wash wells with 100-400 μL of PBS. Collect the PBS and combine it with the supernatant. Add fresh culture medium containing the same concentration of TCE to the same well.

[0064] 6. The number of T cells in each sample was quantified by performing a round-pass cytometry to count the number of cells with fluorescent signals.

[0065] Repeated kill test 1. In a 24-well plate, seed 60,000-240,000 cancer cells expressing the target biomarker into each well.

[0066] 2. PBMCs and 1-2000 ng / mL TCE were added to cancer cells at an effector cell to target ratio of 4:1. The cells were incubated at 37°C and 5% CO2 for up to 48 hours.

[0067] 3. Collect PBMCs. Maintain PBMCs at a cell density of 5e5-3e6 cells / mL in RPMI medium containing 400 U / mL IL-2. Incubate at 37°C and 5% CO2 for up to one week.

[0068] 4. In a 96-well plate, seed 10,000-40,000 cancer cells per well, using the same cancer cells during the first round of co-culture.

[0069] 5. Add activated PBMCs at a series of effector cell to target ratios (maximum 4:1). Incubate at 37°C and 5% CO2 for up to 24 hours.

[0070] 6. Assess target cell viability.

[0071] Proliferation assay 1. In a 24-well plate, seed 60,000-240,000 cancer cells expressing the target biomarker into each well.

[0072] 2. PBMCs were stained with fluorescent cell tracers. The stained PBMCs and 1-2000 ng / mL TCE were added to the cancer cells at an effector cell to target ratio of 4:1. The cells were incubated at 37°C and 5% CO2 for up to 48 hours.

[0073] 3. Collect PBMCs. Maintain PBMCs at a cell density of 5e5-3e6 cells / mL in RPMI medium containing 400 U / mL IL-2. Incubate at 37°C and 5% CO2 for up to one week.

[0074] Cell proliferation was assessed by performing a round of flow cytometry to quantify the number of live PBMCs and the average fluorescence signal carried by the live PBMCs.

[0075] The TCE assay system can be used to screen, identify, and characterize TCEs as candidate therapeutic agents. Furthermore, by using other types of immune cells, including but not limited to natural killer (NK) cells, natural killer T cells (NKT), dendritic cells (DCs), and B lymphocytes (B), the assay system disclosed herein can be used to screen, identify, and characterize other types of conjugates.

[0076] References: 1. Ambrosetti, F., Olsen, TH, Olimpieri, PP, Jiménez-García, B., Milanetti, E., Marcatilli, P., & Bonvin, AMJJ (2020). proABC-2: PRediction of AntiBody contacts v2 and its application to information-drivendocking. Bioinformatics (Oxford, England) , 36 (20), 5107–5108. https: / / doi.org / 10.1093 / bioinformatics / btaa644 2. Chen, X., Zaro, JL, & Shen, WC (2013). Fusion protein linkers: property, design and functionality. Advanced drug delivery reviews , 65 (10), 1357–1369. https: / / doi.org / 10.1016 / j.addr.2012.09.039 sheet Table 1 summarizes the characterization of ARB202 and its CD3 variants and adaptor variants for comparison, including CD3 / CDH17 binding EC50, binding half-life, dissociation half-life, cytotoxic EC50, and IFN-γ EC50, as measured by fold differences.

Claims

1. An assay system for evaluating the efficacy and safety of T-cell engagers (TCEs), comprising: A cell culture plate comprising at least one chamber for seeding a plurality of cells expressing at least one cancer marker in a cell culture medium at a density of at least 30,000 cells per square centimeter per chamber. The cell culture medium further comprises a plurality of T cells, the number of which does not exceed two-thirds of the number of cells expressing at least one cancer marker, and a TCE at a concentration of at least 1 ng per milliliter of the cell culture medium. The TCE has a first binding specificity for the cancer marker and a second binding specificity for the T cells.

2. An assay system for evaluating the efficacy of T-cell engagers (TCEs), comprising: A first cell culture plate, comprising at least one chamber for seeding a plurality of cells expressing at least one cancer marker in a first cell culture medium, at a density of at least 30,000 cells per square centimeter per chamber. The first cell culture medium further comprises a plurality of T cells, the number of which is four times the number of cells expressing at least one cancer marker, and a TCE at a concentration of at least 1 ng per milliliter of the first cell culture medium. The TCE has a first binding specificity for the cancer marker and a second binding specificity for the T cells, and The cells expressing at least one cancer marker, the T cells, and the TCE are co-cultured for a period of time to provide activated T cells.

3. The assay system of claim 2, wherein the TCE is configured to bind the T cells and the cells expressing at least one cancer marker to provide a biological complex thereby activating the T cells, killing the cells expressing at least one cancer marker, and detaching the cells expressing at least one cancer marker from the first cell culture plate.

4. The assay system according to claim 2 further includes a second cell culture plate for measuring the growth rate of the activated T cells. The second cell culture plate contains a second cell culture medium. The activated T cells are removed from the first cell culture medium and cultured in the second cell culture medium for an expansion period to provide expanded activated T cells. The growth rate was measured by pre-labeling the T cells with a fluorescent dye and counting the number of the expanded activated T cells.

5. The assay system according to claim 2 further includes a third cell culture plate for measuring the cytotoxicity of the activated T cells. The third cell culture plate contains cells expressing at least one cancer marker in a third cell culture medium. The activated T cells are removed from the second cell culture medium and co-grown with the cells expressing at least one cancer marker in the third cell culture medium for a co-existing period. The cytotoxicity of the activated T cells is measured by counting cell deaths in the cells expressing at least one cancer marker.

6. The assay system according to claim 1 or 2, wherein the cells expressing the cancer markers include cancer cells.

7. The assay system according to claim 1 or 2, wherein the cancer biomarker comprises CDH17.

8. The assay system according to claim 6, wherein the cancer cells include gastrointestinal cancer cells, gastric cancer cells, colon cancer cells, esophageal cancer cells, pancreatic cancer cells, liver cancer cells, or combinations thereof.

9. The assay system according to claim 1 or 2, wherein the T cells include T lymphocytes derived from peripheral blood mononuclear cells, cytotoxic T cells, helper T cells, or combinations thereof.

10. The system according to claim 1 or 2, wherein the T cells express CD3.

11. The assay system according to claim 1 or 2, wherein the TCE comprises a bispecific antibody or a multispecific antibody.

12. The assay system according to claim 11, wherein the bispecific antibody has binding specificity for CD3 and CDH17.

13. The biological complex of claim 3, comprising the TCE that binds to the T cells and the cells expressing at least one cancer marker.

14. The biological complex of claim 13, wherein the cell expressing at least one cancer marker is a cancer cell.

15. The biological complex of claim 14, wherein the cancer cells are gastrointestinal (GI) cancer cells.

16. A method for evaluating the efficacy and safety of T-cell engagers (TCEs), comprising the following steps: Multiple cells expressing at least one cancer biomarker were attached to a cell culture plate; Multiple T cells were introduced into the cell culture medium; TCE is introduced into the cell culture medium, wherein the TCE has a first binding specificity for the cancer marker and a second binding specificity for the T cells, wherein the TCE is configured to bind the T cells and the cells expressing at least one cancer marker. as well as The rate at which the T cells attach to or detach from the cells expressing at least one cancer marker is measured.

17. A method for evaluating the efficacy and safety of T-cell engagers (TCEs), comprising the following steps: Multiple cells expressing at least one cancer marker are attached to a first cell culture plate and the cells are cultured in a first cell culture medium for a period of time. Multiple T cells were introduced into the first cell culture medium; TCE is introduced into the first cell culture medium, wherein the TCE has a first binding specificity for the cancer marker and a second binding specificity for the T cells, wherein the TCE is configured to bind the T cells and the cells expressing at least one cancer marker. as well as The T cells, which are expressed by at least one cancer marker, are co-cultured with the TCE and attached to the first cell culture plate.

18. The method of claim 17, further comprising collecting T cells detached from the first cell culture plate to provide activated T cells.

19. The method of claim 18, further comprising the step of: The activated T cells are grown in a second cell culture plate containing a second cell culture medium for an expansion culture period to provide expanded activated T cells. The T cells were pre-labeled with a fluorescent dye, and The number of the expanded activated T cells is counted to provide the T cell growth rate.

20. The method of claim 18, further comprising the step of: The activated T cells are grown in a third cell culture plate containing cells expressing at least one cancer marker in a third cell culture medium for a period of co-existence. as well as The cytotoxicity of the activated T cells is measured by counting cell deaths in cells expressing at least one cancer marker.

21. The method according to claims 16 and 17, wherein the TCE is a bispecific antibody with affinity for CD3 and CDH17.

22. The method of claims 16 and 17, wherein the cell expressing the cancer marker is a cancer cell.

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