阅读说明：本技术 用于确定抗cd26配体的生物活性的定量细胞方法 (Quantitative cellular method for determining biological activity of anti-CD 26 ligand ) 是由 A·F·迪纳罗 于 2018-12-21 设计创作，主要内容包括：本发明涉及定量细胞方法,其用于体外确定抗CD26配体(优选抗CD26单克隆抗体(例如,倍格罗单抗)的作用。(The present invention relates to a quantitative cellular method for determining the effect of an anti-CD 26 ligand, preferably an anti-CD 26 monoclonal antibody (e.g., gemuzumab), in vitro.)

1.A method for determining the efficacy of an anti-CD 26 ligand in vitro, comprising the steps of:

a) incubating a population of human T lymphocytes expressing CD26 receptor at a percentage higher than 75% with an anti-CD 26 ligand at a concentration in the range of 0.001 μ g/ml to 150 μ g/ml at 37 ℃ or at room temperature;

b) incubating with an anti-CD 26 antibody labeled with a fluorescent dye, which antibody recognizes a CD26 epitope different from the CD26 epitope recognized by the anti-CD 26 ligand used in step a);

c) determination of the MFI value (MFI) of CD26 measured on cell samples treated with anti-CD 26 ligand by cytofluorimetric analysis_T) And MFI value (MFI) of untreated cells_NT)；

d) Percent internalization (% int CD26) or RFI of CD26 receptor was evaluated by calculation according to the formula:

wherein if the value of% intCD 26:

-less than 20%, indicating low potency against CD26 ligand;

in the range of 20% to 30%, indicating moderate potency against CD26 ligand;

-above 30%, indicating high potency against CD26 ligand.

2. The method according to claim 1, wherein the concentration of the anti-CD 26 ligand in step a) is in the range of 0.01 to 100 μ g/ml, more preferably in the range of 0.01 to 2 μ g/ml, even more preferably in the range of 0.01 to 0.5 μ g/ml.

3. The method of any one of claims 1-2, wherein the anti-CD 26 ligand in step a) is an antibody or fragment thereof.

4. The method of claim 3, wherein the anti-CD 26 antibody is monoclonal antibody bigemumab.

5. The method of any one of the preceding claims, wherein the fluorescent dye in step b) is selected from the group consisting of: FITC, APC, PE-Cy7, APC-H7, PerCP and PE-Cy5.5, preferably APC.

6. The method according to any one of the preceding claims, wherein the population of human CD26+ T lymphocytes in step a) is selected from the group consisting of a primary population of T lymphocytes and a tumor cell line of human T lymphocytes.

7. The method of claim 6, wherein the tumor cell line of human T lymphocytes is the Karpas299 cell line.

8. The method of any one of the preceding claims, wherein the cellular fluorescence analysis in step c) is performed by FACS.

9. The method of any one of the preceding claims, further comprising an assay step of inhibiting the production of an inflammatory cytokine selected from the group consisting of I L-8, I L-1 β, I L-6, I L-2, GM-CSF, I L-6, and TNF- α on the population of human CD26+ T lymphocytes in step a).

10. The method of claim 9, wherein the inhibition of cytokine production is assessed by a mesoscale discovery assay.

11. The method of claim 9 or 10, wherein the population of human CD26+ T lymphocytes is the Karpas299 cell line.

Technical Field

The present invention relates to quantitative cellular methods for determining the effect of an anti-CD 26 ligand, preferably an anti-CD 26 monoclonal antibody (e.g., begallomab), in vitro.

Background

CD26 is a 110kDa multifunctional glycoprotein expressed on the cell surface and in soluble form CD26 antigen is expressed by various tissues and organs (e.g. lung, endothelium, heart, brain, liver, intestine, kidney, placenta, pancreas and skeletal muscle) (abbott c.a. et al, 1994). at the cellular level, expression of CD26 has been found to have strong co-stimulatory activity in lymphocyte populations (in particular in activated T lymphocytes, in resting T lymphocytes and on B lymphocytes) (Cordero OJ et al, 2007). indeed, on a specific subset of memory T cells expression of CD26 is increased upon self-activation of T cells (morimoto c. et al, 1989.) expression of CD26 on T cells is related to the ability of these cells to produce large amounts of I L-2 and to proliferate strongly in response to mitogenic stimuli.

CD26 is characterized by dipeptidyl peptidase IV (DPP-IV) enzymatic activity. The enzymatic activity described above specifically promotes the hydrolysis of the peptide bond between the N-terminal amino acid and the adjacent amino acid at the X-Pro position (Gorrel m.d. et al, 2001). CD26 belongs to a subset of oligopeptidases that are capable of cleaving N-terminal dipeptides from a number of biologically active substrates (e.g., cytokines, polypeptides, hormones and chemokines) (De Meister I. et al, 1999; Hildebrandt M. et al, 2000).

In humans, CD26 is also involved in binding to Adenosine Deaminase (ADA) (Franco r. et al, 1998). ADA deficiency is predisposed to immunodeficiency disease not only by the general mechanism of immune dysregulation but also by the intracellular accumulation of toxic metabolites of purine metabolism (Sauer a.v. et al, 2012).

The possible role of the link between CD26 and ADA is in fact to regulate local extracellular adenosine concentrations, which produce negative signals in T cells via adenosine receptors on the cell surface. Some monoclonal antibodies specific for CD26 are capable of transmitting activation signals to T cells and modulating immune responses in vitro (Morimoto c. and Schlossman S.F et al, 1998). Thus, CD26 is involved in the regulation of inflammation, immune endocrine and neurological functions, and in the pathophysiology of AIDS.

Due to the ubiquitous distribution of CD26, many pathological states are associated with altered expression and/or activity of CD26, while altered expression and/or activity of CD26 is associated with the severity of the corresponding pathological condition. These diseases can be divided into at least five categories: autoimmune and inflammatory diseases, hematologic malignancies, neuroendocrine disorders, infectious diseases and solid tumors. Many researchers have observed that the serum enzymatic activity level of CD26 appears to be altered in various immune-mediated diseases (Klemann c et al, 2016). In clinical studies, it has been observed that changes in the expression/activity of CD26 are involved in various autoimmune diseases, e.g., rheumatoid arthritis, multiple sclerosis, type I diabetes, and graft rejection disease (GvHD) in the host.

Multiple studies with highly selective CD26 inhibitors have shown delayed onset of diabetes, decreased insulitis, and increased numbers of regulatory T cells, suggesting an important role for CD26 in immune regulation.

Some reports on patients suffering from rheumatoid arthritis show a correlation between CD26 expression/enzymatic activity, severity of disease and treatment these findings may pave the way for new therapeutic approaches aimed at inhibiting CD26 enzymatic activity furthermore, CD26 expression on the surface of T cells in the blood and cerebrospinal fluid of patients suffering from multiple sclerosis has been found to be higher (ohnima k. et al, 2011) GvHD is the major complication following Hematopoietic Stem Cell Transplantation (HSCT), HSCT is an important therapy for many blood diseases (Ferrara J. L. m. et al, 2009) GvHD can be classified as acute or chronic based on time of onset and is caused by transplantation of naive T cells derived from donor bone marrow, which damage recipient tissue (Henden a. s., hillg. r., 2015.) GvHD represents a severe limitation on the use of bone marrow as a life saving therapy (weniak. 539. a. 2007).

The development of GvHD generally requires three conditions: (1) the bone marrow of the donor must contain immunocompetent cells, (2) the recipient must express tissue antigens that are not present in the donor, and (3) the recipient should not be able to elicit an effective response that would destroy the transplanted cells.

GvHD is characterized by 3 distinct phases 1) in the first phase, tissue is damaged in the host due to radiotherapy and chemotherapy, releasing pro-inflammatory cytokines, e.g., TNF- α and IFN- γ, 2) in the second activation phase, donor alloreactive T cells are activated by the antigen to which the cells presenting the acceptor Antigen (APC) are exposed, 3) finally, cell proliferation occurs with further secretion of cytokines produced by both cytotoxic T cells and effector T cells (FerraraJ. L. m. et al, 2009).

The pathology, severity and organ specificity of acute gvhd (agvhd) is determined by the balance between different T lymphocyte subsets Th1, Th2 and Th 17. Indeed, it has been shown that the prevalence of the Th1 subtype relative to other subtypes depends on the cytokine environment at the time of donor T cell activation, followed by interaction with the host's APC cells and their subsequent differentiation into helper T cells.

The major Th1 cytokines are IFN- γ, I L-2, and TNF α because of the ability of Th1 cytokines (e.g., TNF and IFN- γ) to induce the upregulation of chemokines and receptors capable of promoting inflammatory responses, increased amounts of Th1 cytokines (e.g., TNF and IFN- γ) have been associated with earlier and more severe onset of disease Th2 cytokines are I L-4, I L-5, I L-10, and I L-13. blockade of responses by Th2 cells has been observed to be associated with increased gastrointestinal symptoms and decreased levels of liver and skin damage.i L-6 is a pro-inflammatory cytokine that controls the balance between Th17 cells and regulatory T cells.i L-6 inhibition may be a potentially effective strategy to reduce the severity of aGvHD in HCT by inducing immune tolerance (Henden a.s., hig.r.

Thus, elucidation of the role of CD26 in T cell responses and altered cytokine levels would undoubtedly help to understand the pathogenesis and progression of GvHD (Henden A.S., Hill G.R., 2015; Yi T.2009).

Based on this premise, a new therapeutic approach to prevent the onset of GvHD is based on the use of an anti-CD 26 monoclonal antibody (Hatano r., 2013; Bacigalupo a., 2016). anti-CD 26 antibodies have recently been developed in preclinical models to prevent the onset of GvHD in animal models of disease. Although the role of CD26/DPPIV in GvHD should be further investigated, treatment with a mouse antibody against human CD26 (i.e., gemuzumab) has been reported to be effective in treating GvHD in patients suffering from acute steroid resistant GvHD (us patent 9376498). Thus, clinical data demonstrate that inactivation of CD26 by monoclonal antibodies represents an effective therapeutic approach for eradicating autoreactive T cell subsets, thereby addressing GVHD.

In view of the above, the importance of the CD26 receptor as a molecular target in new therapeutic approaches is evident.

Potency testing provides a quantitative measure of the biological activity of a particular drug and represents a key quality parameter in the development of pharmacological molecules.

Various methods can be used to develop potency assays, including ligand-receptor assays, animal assays, in vitro cellular assays, or other biochemical assays (e.g., enzymatic assays). A potency test is particularly relevant if it reproduces the mechanism of action of a particular drug.

For biological products, it is preferred to use ligand-receptor assays or in vitro cell assays. The former can also be useful in potency testing because it can provide a direct measure of the affinity of a drug for its molecular target.

However, it is not always possible to use these types of assays, simply because efficacy tests should be developed based on the mechanism of action of the drug, but this information is not always available, especially for monoclonal antibodies. This approach is particularly complex, since biopharmaceuticals often have multiple mechanisms of action in vivo, so that they are particularly difficult to reproduce in an in vitro system.

The authors of the present invention have now found that an anti-CD 26 monoclonal antibody (bigemumab) is capable of inducing internalization of the CD26 receptor after specific binding, with a mechanism of action known as "blocking". The internalization phenomenon results in the inhibition of the release of pro-inflammatory cytokines (critical in the inflammatory process), a direct downstream functional event induced by anti-CD 26 ligands. The mechanism of action described therefore supports all the applications of monoclonal antibodies in all autoimmune diseases, where it is necessary to inactivate autoreactive T lymphocytes while maintaining their immunological activity.

Thus, since efficacy testing of pharmaceutical products is performed in order to determine the amount of active compound in a functional manner in a sample, both internalization of CD26 and inhibition of inflammatory cytokine secretion represent a new test that can be used to assess the efficacy of any anti-CD 26 ligand, preferably to develop monoclonal antibodies for therapeutic and/or diagnostic applications.

Based on the revolutionary discovery of the mechanism of action of gemuzumab, the described method can be applied to assess the efficacy of any anti-CD 26. Furthermore, the use of this method allows to obtain a quantitative measure of both the internalization of CD26 and the inhibition of the secretion/production of cytokines.

In summary, these findings allow the preparation of extremely advantageous potency tests in 1) reproducibility, 2) ease of quantifying the activity of anti-CD 26 ligand in a particular sample, and 3) quantitative measurement of the activity of anti-CD 26 ligand (whether known or newly identified as bigemumab).

Disclosure of Invention

Accordingly, the present invention relates to a method for determining the efficacy of an anti-CD 26 ligand in vitro, comprising the steps of:

a) incubating a population of human T lymphocytes expressing CD26 receptor at a percentage higher than 75% with an anti-CD 26 ligand at a concentration in the range of 0.001 to 150 μ g/ml (preferably 0.01 to 100 μ g/ml, more preferably 0.01 to 2 μ g/ml, even more preferably 0.01 to 0.5 μ g/ml) at 37 ℃;

b) incubating with an anti-CD 26 antibody labeled with a fluorescent dye, said antibody recognizing an epitope on the CD26 receptor that is different from the epitope recognized by the anti-CD 26 ligand of step a);

c) determination of the MFI value (median fluorescence intensity) (MFI) of CD26 measured on cell samples treated with anti-CD 26 ligand by cytofluorescence analysis_T) And MFI value (MFI) of untreated cells_NT)；

d) The percent internalization of the CD26 receptor was assessed as RFI (relative fluorescence intensity, i.e., MFI value normalized to the base), which was calculated according to the following formula: MFI value (MFI) of CD26 measured on cell samples treated with anti-CD 26 ligand_T) MFI value (MFI) with untreated cells_NT) The ratio between, multiplied by 100, and subtracted from 100:

where "% int CD 26" or RFI is the internalization percentage of CD26, "MFI_T"is the MFI value of cells treated with CD26 ligand (test cells), and" MFI_NT"is the MFI value for cells not treated with CD26 ligand (reference cells).

Such percentages (% int CD26 or RFI):

-if less than 20%, indicating low potency against CD26 ligand;

-if in the range of 20% to 30%, indicating moderate potency against CD26 ligand;

if higher than 30%, indicating a high potency against the CD26 ligand.

In panel a of fig. 12, the rationality for specifying "low", "medium" and "high" efficacy criteria, respectively, is indicated. In particular, the response expressed as% internalization of CD26 was observed to be dose-dependent S-shaped (sigmoid) with lower "% int CD 26" values at lower ligand concentrations (denoted "RS" in this figure) and vice versa with greater "% int CD 26" values up to a maximum (plateau) of 30% -35%. If this plateau value is considered to be 100% of "% int CD 26", then all values of the curve can be normalized, as shown in panel B of FIG. 12.

In this sense, a curve starting from 0% to 100% internalization is observed and therefore three different ranges of potency can be defined:

"Low", which means that the ligand has a percent internalization of 0% to 50% relative to the stated plateau value,

"moderate", this means that the ligand has a percent internalization of 50% to 90% relative to the stated plateau value,

"high", which means that the ligand has a percent internalization higher than 90% with respect to the plateau value.

In other words, this concept can be summarized in table 1 below:

TABLE 1

Efficacy	RFI	% plateau value
			Is low in	0％-20％	0％-50％
Medium and high grade	20％-30％	50％-90％
			Height of	>30％	90％-100％

In an alternative embodiment of the invention, the population of human lymphocytes of step a) can be incubated at room temperature.

The anti-CD 26 ligand is any molecule capable of specifically binding to the CD26 receptor, preferably an anti-CD 26 monoclonal antibody or fragment thereof, more preferably gemtuzumab.

In a preferred embodiment of the method of the invention, the concentration of the anti-CD 26 ligand in step a) is 0.001. mu.g/ml, 0.01. mu.g/ml, 0.5. mu.g/ml or 2. mu.g/ml.

In a preferred embodiment of the method of the invention, said anti-CD 26 antibody in step b) is a mouse anti-human CD26 APC fluorochrome-conjugated type (BD Pharmingen; catalog number: 563670, clone number: M-A261) and incubated at a concentration of 2.5. mu.g/ml.

According to another preferred embodiment of the method of the invention, the fluorescent dye used in step b) is any fluorescent dye that can be used for fluorescence analysis of cells, selected from the group consisting of: FITC, APC, PE-Cy7, APC-H7, PerCP, and PE-Cy5.5. The fluorescent dye is preferably APC.

Likewise, according to a preferred embodiment of the method of the invention, said population of CD26+ T lymphocytes in step a) is selected from the group consisting of a primary T lymphocyte population and a tumor cell line of human T lymphocytes. Preferably, the tumor cell line of human T lymphocytes is the Karpas299 cell line.

In a preferred embodiment of the method according to the invention, the cellular fluorescence analysis in step c) is performed by FACS.

The method according to the invention also provides a further validation step of the potency of an anti-CD 26 ligand against the CD26+ human T lymphocyte population in step a), which provides the above-mentioned ligand inhibition test for the release of a cytokine selected from the group consisting of I L-8, I L-1 β, I L-6, I L-2, GM-CSF, I L-6 and TNF- α in a preferred embodiment the cytokine is I L-8 and/or I L-1 β in step a) the CD26+ human T lymphocyte population is selected from the group of primary T lymphocytes and the tumor cell line of human T lymphocytes.

In a preferred embodiment, the cytokine production inhibition test is performed by a mesoscale discovery (msd) assay.

Drawings

The present invention will now be described, for illustrative and non-limiting purposes, according to a preferred embodiment with particular reference to the accompanying drawings, in which:

figure 1 shows a cell culture protocol for analysis of proinflammatory cytokine levels using the MSD assay.

FIG. 2 shows the cytofluorescence analysis of the percentages of CD4+ cells (dark bars) and CD26+ cells (light bars) in peripheral blood mononuclear cells and purified CD4+ T cells (A) and the human T cell line Karpas299 (B).

Figure 3 shows the effect of gemtuzumab on CD26 receptor internalization after 8 hours of incubation in Karpas299 cells (panel a) and after 6 hours and 24 hours of incubation in primary CD3+, CD4+, CD8+ T cells at 4 ℃ and 37 ℃ (panel B, C, D).

FIG. 4 shows the inhibition of the level of cytokines I L-2, I L-8, I L-1 β, GM-CSF, I L-6 and TNF- α induced by gemuzumab in the T cell line Karpas299 (panel A) and in primary T cells (panel B). in this figure, the electrochemiluminescence signal is indicated on the left of the ordinate axis, while the corresponding concentration is indicated on the abscissa axis.

Figure 5 shows the decrease in fluorescence associated with CD26 after 24 hours of treatment with gemuzumab in Karpas299 cells.

FIG. 6 shows that from 0.0005. mu.g/m L (3X 10)^-12M) until a higher concentration equal to 150. mu.g/M L (1X 10)^-6M) was reduced in fluorescence associated with CD26 after 24 hours in Karpas299 cells.

Figure 7 shows the dose-response internalization curve (RFI) of CD26 obtained by FACS analysis of samples of gemtuzumab stored under different temperature conditions (SVI-STB is a sample with reduced potency, i.e. the sample is left for 6 months under accelerated stabilization conditions at 32.5 ℃).

Figure 8 shows the CD26 internalization curves (RFI) obtained by FACS analysis performed on different cytofluorimeters.

Figure 9 shows the relative potency of the mouse monoclonal anti-hCD 26 antibody (clone 202.36) observed against gemtuzumab (WS-BEG-013).

FIG. 10 shows the relative potency of IgG2b mouse antibody K isotype (clone MG2b-57) observed relative to gemolizumab (WS-BEG-013).

Figure 11 shows the potency values of the TEST samples (TEST) calculated with respect to the reference standard RS (gemuzumab) to demonstrate how the relative activity of any unknown sample named TEST is quantitatively measured with respect to the Reference Standard (RS).

Fig. 12 shows two exemplary diagrams for determining criteria for low, medium or high forces.

Detailed Description

In order to better illustrate the invention, the following examples are now provided, which should be considered as illustrative and non-limiting.