阅读说明：本技术 改进的免疫原性测定法 (Improved immunogenicity assays ) 是由 安内利斯·科登斯 维尔勒·斯诺尔克 玛丽-安格·比斯 卡罗·布东 于 2018-03-29 设计创作，主要内容包括：本发明涉及改进的免疫原性测定和用于进行该测定的方法。特别地,本发明涉及能够区分中和性抗药物抗体和非中和性抗药物抗体的改进的免疫原性测定。更特别地,本发明涉及改进的免疫原性测定,其允许在样品中检测和/或测定中和性抗药物抗体(的量和/或浓度),甚至在存在非中和性抗药物抗体的情况下。(The present invention relates to improved immunogenicity assays and methods for performing the assays. In particular, the invention relates to improved immunogenicity assays capable of distinguishing neutralizing anti-drug antibodies from non-neutralizing anti-drug antibodies. More particularly, the present invention relates to an improved immunogenicity assay which allows the detection and/or determination of (the amount and/or concentration of) neutralizing anti-drug antibodies in a sample, even in the presence of non-neutralizing anti-drug antibodies.)

1. A method of detecting and/or assaying an anti-drug antibody (ADA) against an ISV-based drug in a sample, wherein the ISV-based drug comprises one or more Immunoglobulin Single Variable Domains (ISVD), wherein each such ISVD comprises a Framework Region (FR) and a Complementarity Determining Region (CDR), and wherein at least one of the ISVDs present in the ISV-based drug is specific for a therapeutically relevant target, the method comprising performing a bridging anti-drug antibody assay in the presence of a variant of the ISV-based drug that renders it non-functional for binding to the therapeutic target to which the ISV-based drug is to bind (but otherwise substantially identical to the ISV-based drug).

2. The method of claim 1 wherein a bridging anti-drug antibody assay is performed in the presence of a variant of the ISV-based drug that renders it unable to specifically bind to the therapeutic target to which the ISV-based drug is to bind (but otherwise is substantially identical to the ISV-based drug).

3. The method of claim 1 or 2, wherein a bridging anti-drug antibody assay is performed in the presence of a variant of the ISV-based drug, wherein the variant is substantially identical to the ISV-based drug except that one or more ISVs in the variant that correspond to the one or more ISVs in the ISV-based drug that are intended to bind the therapeutic target(s) of the ISV-based drug have CDRs that are different from CDRs in corresponding ISVs in the ISV-based drug such that the variant is non-functional for binding to the therapeutic target to which the ISV-based drug is to bind (but otherwise substantially identical to the ISV-based drug).

4. The method of any one of claims 1-3, wherein a bridging anti-drug antibody assay is performed in the presence of a variant of the ISV-based drug, wherein the variant is substantially identical to the ISV-based drug except that one or more ISVs in a variant that correspond to the one or more ISVs in the ISV-based drug that are intended to bind the ISV-based drug have CDRs that are different from CDRs in corresponding ISVs in an ISV-based drug, such that the variant is unable to specifically bind (as defined herein) the therapeutic target to which the ISV-based drug is to bind (but otherwise is substantially identical to the ISV-based drug).

5. The method of any one of the preceding claims, wherein the variant has substantially the same amino acid sequence as the ISV-based drug except that the alteration of the CDRs in the variant removes the ability of an ISV present in the variant to bind to a therapeutic target of the ISV-based drug.

6. The method of any one of the preceding claims, wherein the variant has a degree of sequence identity (as defined herein) with the ISV-based drug of at least 90%, such as at least 95%, particularly at least 99% (up to and including 100%), irrespective of changes in CDRs which remove the ability of an ISV present in the variant to bind to a therapeutic target of the ISV-based drug.

7. The method of any one of the preceding claims, wherein performing the bridging anti-drug antibody assay comprises the steps of:

a) Contacting the sample with a capture agent immobilized on a support under conditions such that any anti-drug antibodies to the ISV-based drug can bind to the capture agent, wherein the capture agent is or consists essentially of the ISV-based drug;

b) (optionally) removing any components or constituents present in the sample that are not bound to the capture agent;

c) Any anti-drug antibody that has bound to the capture agent is detected or assayed by contacting the complex of capture agent and any captured anti-drug antibody with a detection agent that is or consists essentially of the ISV-based drug linked to a detectable label under conditions such that the detection agent can bind to (the complex of capture agent and) any captured anti-drug antibody.

8. The method of claim 7, wherein in step c) substantially only neutralizing ADA for the ISV-based drug is detected or measured.

9. the method of any one of the preceding claims, which is performed on a sample containing (or suspected of containing) neutralizing and non-neutralizing ADA against the ISV-based drug.

10. The method of any one of the preceding claims, which is performed on a sample obtained from a human subject.

11. The method according to any one of the preceding claims, which is performed on a sample of whole blood, serum, plasma, lymph, ocular fluid, bronchoalveolar fluid/BALF, cerebrospinal fluid or another biological fluid (e.g. sputum or nasal wash); in particular a sample of whole blood, serum or plasma.

12. The method of any one of the preceding claims, wherein the ISV-based drug has a half-life in humans of at least 3 days (expressed as t1/2 β).

13. The method of any one of the preceding claims, wherein the ISV-based medicament comprises at least two ISVs, such as 2, 3, 4, or 5 ISVs.

14. The method of claim 13, wherein the ISV-based drug comprises at least one ISV against serum albumin.

example 1

As a first example, the Competitive Ligand Binding Assay (CLBA) of the present invention was used, which was developed with the following requirements: (i) drug tolerance: a predicted maximum drug level of 30 μ g/mL; (ii) target tolerance: a predicted maximum target level of 1500 ng/mL; and (iii) sensitivity: 250-500ng/mL positive control antibody in the absence of drug (see Table I). The sample is subjected to a pre-treatment step substantially as shown in figure 6.

Assay formats were tested using a set of established neutralizing and non-neutralizing abs. The neutralizing potential of monoclonal antibodies (mabs) is based on their ability to block target interactions as determined in CLBA (in buffer without pretreatment). NAb was found to be detectable with the same sensitivity as the ADA assay (see table 2). Non-neutralizing Ab was not detected; some residual binding could be detected at very high Ab concentrations, however these levels are not expected to be clinically relevant.

To further illustrate the effectiveness of the assay method of the present invention, polyclonal rabbit abs were specifically generated by immunization. This polyclonal Ab was used as NAb positive control antibody. Using the assay of the invention, preclinical study samples (rhesus monkeys) from disease models known to be susceptible to ADA development were analyzed (data not shown) and found to contain neutralizing activity by PD and efficacy markers. The results are shown in Table 3. Neutralization scores in ADA positive samples (rabbit pAb and preclinical study samples) can be determined by titer and/or sensitivity assays.

As can be seen from the data in table 4, the assay methods of the invention generally have similar accuracy, sensitivity and drug tolerance characteristics compared to conventional ADA assay methods, such as the ADA assay on which the assay of the invention is based. Similar sensitivity and drug tolerance, in compliance with current regulatory guidelines (< 100ng/mL positive control, in the presence of the highest expected drug level).

The data in table 5 show that the target tolerance characteristics are similar to the ADA assay. In the case of monomeric targets, this NAb format is target-tolerant (accuracy between run-and-assay:. ltoreq.20%).

Figure 7 shows the sequences of nanobody constructs and corresponding null variants used to generate the data shown in the above table, using an assay protocol substantially similar to that described in the following paragraphs.

For example, to determine the sequence for SEQ ID NO: 3, NAb of ISV-based drug, can be used SEQ ID NO: 4, e.g. as a detection agent with SEQ ID NO: 3-concentration of 100X concentration of labeled ISV-drug. The assay can be performed as an Electrochemiluminescence (ECL) bridging assay as follows: a master mix consisting of biotinylated, sulfo-labeled drug (bivalent nanobody construct of SEQ ID NO: 3) and excess null variant molecules (SEQ ID NO: 4) was mixed with a mixture of antibodies that may contain a heavy chain variable region directed against SEQ ID NO: 3 of ISV-based drugs. After pre-incubation, the mixture was applied to 96-well streptavidin gold plates. Biotinylated ISV-based drugs in the complex bind to streptavidin on the plate and are detected by sulfo-labeled ISV-based drugs in a Quickplex SQ120 MSD reader after application of current to the plate. An excess of null variants in this assay will capture all non-neutralizing antibodies, including so-called pre-existing antibodies, and enable sensitive detection of neutralizing antibodies. Further assay conditions may for example be as follows:

-Minimum Required Dilution (MRD) 1/100.

Assay dilution buffer: PBS/0.1% casein (prepared from PBS/1% casein, from Biorad)

-MRD: 100 ═ 1% base (including dilution in assay diluent 1/33 followed by dilution in master mix 1/3)

-a master mix: SEQ ID NO: 3, biotinylated ISV-based drug of SEQ ID NO: 3 (ratio 1/1: 2.0 μ g/mL [ SEQ ID NO: 3] -BIO, 2.0 μ g/mL [ SEQ ID NO: 3] -SULFO) and 200 μ g/mL of the peptide of SEQ ID NO: 4 null variants

-reading buffer: read buffer (2 ×) (diluted in MQ)

TABLE 1