WO2023094696A1

WO2023094696A1 - Methods for reducing anti-cd38 mab drug interference in serological assays

Info

Publication number: WO2023094696A1
Application number: PCT/EP2022/083657
Authority: WO
Inventors: Stefan HÄRTLE; Julia RAUSER; Elke WEINIG
Original assignee: Morphosys Ag
Priority date: 2021-11-29
Filing date: 2022-11-29
Publication date: 2023-06-01
Also published as: TW202328207A

Abstract

The present invention relates to an in vitro method to reduce, prevent or abolish interference of a therapeutic anti-CD38 antibody in serological assay systems.

Description

METHODS FOR REDUCING ANTI-CD38 MAB DRUG INTERFERENCE IN SEROLOGICAL ASSAYS

Field of the Invention

This invention relates to methods and reagents for use in reducing interference in blood bank serological compatibility assays by drugs that comprise (i) an antibody Fc region and (ii) a moiety that binds to human CD38.

Background

CD38 is an integral membrane protein that is highly expressed on myeloma cells and plasma cells and weakly expressed on red blood cells (RBCs). CD38 is an effective target antigen for monoclonal antibody therapies. Approved therapeutic anti-CD38 monoclonal antibody include daratumumab (Darzalex, Janssen Biotech) and isatuximab (Sarclisa, Sanofi). Other CD38 monoclonal antibodies are currently under development.

Drug interference is a well-known phenomenon in laboratory medicine, but can be different for each drug and each analytical method. Therapeutic anti-CD38 monoclonal antibodies may have the potential to interfere with immunohematologic tests systems such as the Indirect Coombs test, also referred to as the indirect antiglobulin test (I AT). This serologic test is used to detect antibodyantigen reactions in vitro, for example to identify low concentrations of antibodies present in a patient's plasma/serum specifically binding to red blood cells (RBCs) of certain blood donors. In more detail, within each IAT serum/plasma collected from the recipient of a blood transfusion is incubated with a wide range of RBCs from different donors that together exhibit a full range of surface antigens (i.e. blood types).

The IAT is a two-stage test. In the first stage, blood donor derived and washed RBCs with known surface antigens are incubated with patient serum containing an unknown composition of antibodies. If the serum contains antibodies to antigens on the RBC surface, the antibodies will bind to the surface of the RBCs. In the second stage, the RBCs are washed with isotonic saline solution and then incubated with antihuman globulin (also known as Coombs reagent). If antibodies have bound to RBC surface antigens in the first stage, RBCs will agglutinate when incubated with the antihuman globulin in the second stage, and the IAT will be positive.

Because CD38 is weakly expressed on RBCs, therapeutic anti-CD38 antibodies may lead to agglutination of RBCs in lATs. It has been observed that plasma samples from anti-CD38- treated patients consistently cause positive reactions in direct antiglobulin tests (DATs), indirect antiglobulin tests (lATs), antibody detection (screening) tests, antibody identification panels, and antihuman globulin (AHG) crossmatches. However, anti-CD38 does not interfere with ABO/RhD typing.

Treatment of patients with an anti-CD38 mAb, resulted in (false) positive lATs for all patients for 2 to 6 months after infusion. As a result, the interference of therapeutic anti-CD38 antibodies in lATs may falsely suggest incompatibility of a suitable blood donor. Such false-positive results precluded the correct identification of irregular blood group antibodies for patients requiring blood transfusion (Oostendorp et al., Transfusion 2015; 55(6pt2): 1555-62.; Chapuy et al., Transfusion 2015;55(6pt2): 1545-54.).

Also some rare Lu(a-b-) cells are not reactive in the presence of anti-CD38 mAb, which may lead to a masking of Lutheran-related antibody potentially present in a patient’s serum/plasma sample (Velliquette RW, Transfusion 2015;55(3S):26A.; Aye T; Transfusion 2015;55(3S):28A.).

In clinical studies, it has been demonstrated that the binding of therapeutic anti-CD38 mAb to erythrocytes does not have any major impact in vivo other than a potentially small non-significant decrease in hemoglobin levels and a compensatory rise in reticulocyte count.

Anti-CD38 interference would primarily mask other potential positive reactions underlying (i.e. the identification of antibodies specifically directed to other antigens than CD38). If the blood bank transfusion service is unaware that a patient has received anti-CD38 therapy, the following scenarios may occur when the patient’s sample is tested:

1. ABO/RhD typing: no issues

2. Antibody detection (screening) test (IAT): several or all cells positive

3. Antibody identification panel: several or all cells positive (autocontrol may be negative)

4. DAT : positive or negative

5. AHG crossmatches: positive with all RBC units tested

6. Adsorptions: panreactivity cannot be eliminated

This leads to delays in issuing RBCs to the patient as the test results suggest the presence of antibodies against various anti-RBC antigen. In some cases, the anti-CD38 interference could mask the presence of a clinically significant alloantibody.

These immunohaematologic issues in in vitro assays incurred by anti-CD38 mAbs are known and several solutions have been proposed (WagnerFF, Blood Transfus 2020; 18: 244-6). The strategy that is usually applied is the use of dithiothreitol (DTT) to pretreat RBCs used in lATs. DTT cleaves the dithio (S-S) bridges of proteins and thereby causes a denaturation of the CD38 antigen on the surface of RBCs. Therefore, an antibody screen can be performed as usual if the RBCs have been treated with DTT. A huge disadvantage of the DTT treatment is that, in addition to CD38, several other antigens on the surface of RBCs are also destroyed, including clinically relevant antigens as Kell (k), Yt^A or the Dombrock (Do) antigens and the method is not always successful. Because of these limitations many alternative methods are proposed as reviewed by Wagner 2020. These methods comprise: (a) The use of trypsin to destroy the CD38 antigen (Chapuy et al., 2015). In contrast to DTT, the K antigen is preserved, but several other blood group antigens like M, N and the Dombrock antigens cannot be detected after trypsin treatment. Furthermore, because trypsin does not completely destroy CD38, trypsin treatment usually fails in patients with high anti-CD38 mAb blood concentrations.

(b) Alternatives to the IAT include, e.g. papainised cells in direct agglutination (Carreno-Tarragona G, et al. Transfus Med 2019) or the polybrene test (Yeh TJ et al. Transfusion 2019; 59: 2751-2). While these alternatives are barely affected by anti-CD38 mAbs, they are not considered an optimal substitute for the IAT because antibodies to important clinical antigens may be missed, like anti-Fy^a using papainised cells or anti-K in the polybrene test.

(c) The use of recombinant soluble CD38 to inhibit anti-CD38 mAbs. Although this approach is feasible, very high concentrations of soluble CD38 are needed.

(d) The use of anti-idiotype antibodies to neutralize the anti-CD38 mAbs. However, specific anti- idiotypic antibodies are difficult to generate, rarely commercially available, and therefore hardly ever used.

(e) The blocking of free CD38 epitopes on RBC by pre-incubation of the RBC with (i) anti-CD38 followed by saturating this anti-CD38 with anti-human globulin, or (ii) Fab-fragment of anti-CD38 or a commercially available Fab2-fragment of anti-CD38 (“DaraEx”) (Selleng K et al. . NEJM, 2019 379;1).

A potential interference of the human anti-CD38 antibody felzartamab (MOR202), currently in clinical studies, in immunohematologic in vitro assays is an emerging concern that requires immediate recognition and the development of appropriate solutions.

Such interference caused by felzartamab (MOR202, TJ202) may be neutralized using erythrocytes pretreated with DTT, soluble CD38-Fc and the anti-idiotypic antibody MOR09292, which is specifically directed against felzartamab.

Summary of the Invention

A novel method is disclosed for the prevention of (false) positive reactions in immunological testing which are caused by interference of a therapeutic human anti-CD38 antibody.

Provided is a method of reducing drug interference in a serological assay using red blood cells (RBC), reagent platelets, or a combination thereof, said method comprising: (a) adding a drug neutralizing agent that binds to the drug and blocks the drug from binding the reagent RBC to a sample from a subject who has received treatment with the drug; and (b) performing the serological assay of the sample after step (a) using the RBC, wherein the drug comprises (i) a human antibody Fc region or variant thereof and (ii) a moiety that binds to human CD38. In some embodiments, the drug is an anti-CD38 antibody. In some embodiments, the drug neutralizing agent is an anti-idiotypic antibody that binds to the antigen binding portion of the anti- CD38 antibody.

Also provided is a method of reducing anti-CD38 mAb interference in a serological assay of a blood sample comprising red blood cells (RBC), platelets, or a combination thereof, said method comprising: (a) adding an anti-idiotypic antibody specific for an anti-CD38 antibody to the blood sample from a subject who has received treatment with an anti-CD38 antibody; and (b) performing the serological assay of the blood sample after step (a), wherein the anti-CD38 antibody comprises an antibody Fc region, and wherein the anti-idiotypic antibody displaces the anti-CD38 antibody bound to CD38 on the surface of the RBC in the blood sample.

In a preferred embodiment, the anti-CD38 antibody is felzartamab (MOR202, TJ202).

In an aspect is an anti-idiotypic antibody to felzartamab (MOR202, TJ202).

In some embodiments, the anti-idiotypic antibody comprises a variable heavy chain comprising an HCDR1 of the amino acid sequence YSFSNYWIS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 2), an HCDR3 of the amino acid sequence SRGAGMDY (SEQ ID NO: 3), and a variable light chain comprising an LCDR1 of the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 4), an LCDR2 of the amino acid sequence LLIYADNNRPS (SEQ ID NO: 5), an LCDR3 of the amino acid sequence GSYDESSNSM (SEQ ID NO: 6).

In an embodiment, the anti-idiotypic antibody is a human antibody.

In some embodiments, the anti-idiotypic antibody specific for an anti-CD38 antibody comprises (a) a heavy chain variable domain (VH) that comprises the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain (VL) that comprises the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8) In some embodiments, the anti-idiotypic antibody comprises the heavy chain of amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT VPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK (SEQ ID NO: 9) and the light chain of amino acid sequence

DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVLGQPKAAPSVTLFPPS SEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQ WKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10).

In a preferred embodiment, the anti-idiotypic antibody is MOR09292.

In some embodiments, the anti-idiotypic antibody is added to the blood sample in a molar excess amount relative to the amount of drug in the blood sample.

In one embodiment, the anti-idiotypic antibody is added to the sample in a molar-excess to the drug of about 2-fold. In a preferred embodiment, MOR09292 is added to the sample in a molarexcess to MOR202 of about 2-fold. In another embodiment, MOR09292 is added to the sample in a 2-fold concentration excess to MOR202.

Also provided is a method of reducing drug interference in a serological assay of a blood sample comprising reagent red blood cells (RBCs), reagent platelets, or a combination thereof, said method comprising: (a) adding a cell binding agent that binds to human CD38 and does not comprise an antibody Fc region to a blood sample from a subject who has received treatment with an anti-CD38 antibody; and (b) performing the serological assay of the blood sample after step (a), wherein the anti-CD38 antibody comprises an antibody Fc region.

In some embodiments, the cell binding agent comprises an antigen-binding fragment of the anti- CD38 antibody MOR202. In some embodiments, the antigen binding fragment is a Fab, a Fab’, a Fab’-SH, an F(ab’)2, an Fv, an scFv, or a diabody. In some embodiments, the serological assay is a direct antiglobulin test (DAT). In some embodiments, the serological assay is an indirect antiglobulin test (IAT).

All references cited herein, including patent applications and patent publications are herein incorporated by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

Figure 1 :

Representative results of negative antibody screening test using the BioRad system.

Figure 2:

Representative results (6 different test erythrocytes) of positive antibody identification due to MOR202 interference using the BioRad system. Plasma sample L#BRH1221755; (A) results shown for MOR202 concentration at 10 pg/mL (B) results shown for MOR202 concentration at 300 pg/mL; arrows indicate visible agglutination.

Figure 3:

Representative results (8 different test erythrocytes) of positive antibody identification due to MOR202 interference using the Grifols system. Plasma sample L#BRH1221755; results shown for MOR202 concentration 30 pg/mL; arrows indicate visible agglutination.

Figure 4:

Representative results (6 different test erythrocytes) of a negative antibody identification testing using MOR202 concentration at 300 pg/mL, RBC pretreatment with 0.2 M DTT, plasma sample L#BRH 1221755.

Figure 5:

Representative results (6 different test erythrocytes) of a positive control antibody identification test despite using DTT pretreatment of RBCs; positive control antibody: alloantibody anti-Fya, MOR202 concentration at 300 pg/mL, RBC pretreatment with 0.2 M DTT, plasma sample L#BRH 1221755.

Figure 6:

Felzartamab at clinically relevant concentrations (here: 300 pg/mL) induces coagulation in several test samples evaluating blood compatibility. 11 blood samples with different erythrocyte antigen combinations (BioRad Diapanel); 12th vial: control without erythrocytes. Figure 7:

The anti-idiotypic mAb MOR09292 neutralizes any coagulation effects induced by felzartamab. Felzartamab 300 pg/mL + MOR09292 600 pg/mL. 11 blood samples with different erythrocyte antigen combinations (BioRad Diapanel); 12th vial: control without erythrocytes.

Figure 8:

In the presence of felzartamab, other coagulating antibodies (here: alloantibody anti-Fy^a) are still able to cause agglutination. 11 blood samples with different erythrocyte antigen combinations (BioRad Diapanel); 12th vial: control without erythrocytes.

Figure 9:

Anti-idiotypic antibody MOR09292 specifically neutralizes felzartamab interference but does not affect other anti-erythrocyte antibodies (i.e. alloantibody anti-Fy^a causes agglutination also in the presence of MOR09292). 11 blood samples with different erythrocyte antigen combinations (BioRad Diapanel); 12th vial: control without erythrocytes.

Detailed Description of the Invention

Definitions

The term “anti-idiotypic” describes a protein or peptide that binds to the variable regions of an antibody. The anti-idiotypic protein can be an antibody. For example, antibody MOR09292 binds to the variable regions of MOR202.

The term "antibody" includes antibody fragments. Antibodies include monoclonal antibodies of any isotype, e.g., IgG, IgM, IgA, IgD and IgE. An IgG antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. The heavy and light chains of antibodies contain a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions", which are primarily responsible for binding an epitope of an antigen. They are referred to as CDR1 , CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions outside of the CDRs are called the "framework regions". An “antibody fragment” means an Fv, scFv, dsFv, Fab, Fab', F(ab')2 fragment, or other fragment, which contains at least one variable heavy or variable light chain, each containing CDRs and framework regions.

The “CDRs” herein are defined by either Chothia et al., Kabat et al. or by an internal numbering convention. See Chothia C, Lesk AM. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol., 196(4):901-17, which is incorporated by reference in its entirety. See Kabat E.A, Wu T.T., Perry H.M., Gottesman K.S. and Foeller C. (1991). Sequences of Proteins of Immunological Interest. 5th edit., NIH Publication no. 91-3242, US Dept, of Health and Human Services, Washington, DC, which is incorporated by reference in its entirety. "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, or antibody fragment. "VL" refers to the variable region of the immunoglobulin light chain of an antibody, or antibody fragment. “Fc region” means the constant region of an antibody, which may be of the lgG1 , 2, 3, 4 subclass or variants thereof.

“Specific” describes a protein that recognizes an antigen and is able to discriminate between such antigen and one or more reference antigen(s). This ability can be identified by a standard ELISA assay. Typically, determination of specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.

The term “CD38” refers to the protein known as CD38. Human CD38 has the amino acid sequence of: MANCEFSPVSGDKPCCRLSRRAQLCLGVSILVLILVVVLAVVVPRWRQQWSGPGTTKRFPETV LARCVKYTEIHPEMRHVDCQSVWDAFKGAFISKHPCNITEEDYQPLMKLGTQTVPCNKILLWS RIKDLAHQFTQVQRDMFTLEDTLLGYLADDLTWCGEFNTSKINYQSCPDWRKDCSNNPVSVF WKTVSRRFAEAACDWHVMLNGSRSKIFDKNSTFGSVEVHNLQPEKVQTLEAWVIHGGREDS RDLCQDPTIKELESIISKRNIQFSCKNIYRPDKFLQCVKNPEDSSCTSEI (SEQ ID NO: 11).

As used herein, the term "biosimilar" is consistent with the working definition promulgated by the United States Food and Drug Administration (FDA) which defines a biosimilar product to be one that is "highly similar" to a reference product (despite minor differences in clinically inactive components). In practice there can be no clinically meaningful differences between the reference product and the biosimilar product in terms of safety, purity, and potency (Public Health Service (PHS) Act §262). The "reference product" refers, for example, to commercially available felzartamab.

“Felzartamab” is an anti-CD38 antibody, also known as “MOR202”, TJ202, “MOR03087” or “MOR3087”. The terms are used interchangeable in the present disclosure. MOR202 has an I gG 1 Fc region.

The amino acid sequence of the MOR202 HCDR1 according to Kabat is: SYYMN (SEQ ID NO: 12).

The amino acid sequence of the MOR202 HCDR2 according to Kabat is: GISGDPSNTYYADSVKG (SEQ ID NO: 13).

The amino acid sequence of the MQR202 HCDR3 according to Kabat is: DLPLVYTGFAY (SEQ ID NO: 14).

The amino acid sequence of the MQR202 LCDR1 according to Kabat is: SGDNLRHYYVY (SEQ ID NO: 15). The amino acid sequence of the MQR202 LCDR2 according to Kabat is: GDSKRPS (SEQ ID NO: 16).

The amino acid sequence of the MOR202 LCDR3 is: QTYTGGASL (SEQ ID NO: 17).

The amino acid sequence of the MOR202 heavy chain variable domain (VH) is: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS (SEQ ID NO: 18).

The amino acid sequence of the MQR202 light chain variable domain (VL) is: DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSG SNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 19).

The antibody MQR09292 is an anti-idiotypic antibody to MQR202.

MQR09292, the anti-idiotypic antibody specific for the anti-CD38 antibody MQR202 comprises

(a) a heavy chain variable domain (VH) that comprises the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain (VL) that comprises the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8).

The heavy chain variable domain of MQR09292 is encoded by the nucleic acid sequence: CAGGTGCAATTGGTTCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGAAAGCCTGAAAATT AGCTGCAAAGGTTCCGGATATTCCTTTTCTAATTATTGGATTTCTTGGGTGCGCCAGATGC CTGGGAAGGGTCTCGAGTGGATGGGCATTATCGATCCGGCTTCTAGCAAGACCCGTTATT CTCCGAGCTTTCAGGGCCAGGTGACCATTAGCGCGGATAAAAGCATTAGCACCGCGTATC TTCAATGGAGCAGCCTGAAAGCGAGCGATACGGCCATGTATTATTGCGCGCGTTCTCGTG GTGCTGGTATGGATTATTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 20).

The light chain variable domain of MQR09292 is encoded by the nucleic acid sequence: GATATCGTGCTGACCCAGCCGCCTTCAGTGAGTGGCGCACCAGGTCAGCGTGTGACCATC TCGTGTACGGGCAGCAGCAGCAACATTGGTGCTGGTTATGATGTGCATTGGTACCAGCAG

TTGCCCGGGACGGCGCCGAAACTTCTGATTTATGCTGATAATAATCGTCCCTCAGGCGTG CCGGATCGTTTTAGCGGATCCAAAAGCGGCACCAGCGCGAGCCTTGCGATTACGGGCCT GCAAAGCGAAGACGAAGCGGATTATTATTGCGGTTCTTATGATGAGTCTTCTAATTCTATG

GTGTTTGGCGGCGGCACGAAGTTAACCGTTCTTGGCCAG (SEQ ID NO: 21).

Embodiments

In some embodiments, the method comprises (a) adding an anti-idiotypic antibody that binds a therapeutic anti-CD38 antibody (i.e. to the portion of the therapeutic anti-CD38 antibody that comprises the moiety that binds to human CD38) to a biological sample from a subject who has received treatment with the anti-CD38 antibody, and (b) performing the serological assay of the plasma sample after step (a) using RBCs (i.e., RBCs that are known to express a particular cell surface antigen, or group of cell surface antigens) and/or platelets (i.e., platelets that are known to express a particular cell surface antigen, or group of cell surface antigens), wherein the anti- CD38 antibody comprises an Fc region.

The anti-idiotypic antibody binds to the therapeutic anti-CD38 antibody (e.g., to the moiety that binds to human CD38) in the subject’s biological sample and blocks the anti-CD38 antibody from binding the reagent RBCs and/or reagent platelets. The interference that would result from the binding of anti-CD38 antibody to the RBCs and/or platelets is minimized (or, in some embodiments, eliminated), thus preventing a false positive result in the serological assay. In some embodiments, the anti-idiotypic antibody specific for the therapeutic anti-CD38 antibody is also added to the RBCs and/or platelets before the serological assay is performed.

In one embodiment, the biological sample from a subject is serum or plasma from said subject.

In one aspect, a method is provided to mitigate, reduce or abolish interference of an anti-CD38 antibody drug in in vitro serological assay systems comprising red blood cells and/or platelets, wherein the method comprises the following steps:

(a) adding a drug neutralizing agent to a serum or plasma sample from a subject who has received treatment with the drug; and

(b) performing the serological assay of the serum or plasma sample after step (a), using red blood cells and/or the platelets, wherein the anti-CD38 antibody drug comprises a human antibody Fc region or variant thereof.

In a further aspect, the anti-CD38 antibody drug comprises an HCDR1 of the amino acid sequence SYYMN (SEQ ID NO: 12), an HCDR2 of the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO: 13), an HCDR3 of the amino acid sequence DLPLVYTGFAY (SEQ ID NO: 14) an LCDR1 of the amino acid sequence: SGDNLRHYYVY (SEQ ID NO: 15), an LCDR2 of the amino acid sequence: GDSKRPS (SEQ ID NO: 16), and an LCDR3 of the amino acid sequence: QTYTGGASL (SEQ ID NO: 17).

In one embodiment, the anti-CD38 antibody drug comprises a heavy chain variable domain (VH) of the amino acid sequence:

QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS (SEQ ID NO: 18) and a light chain variable domain (VL) of the amino acid sequence:

DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSG SNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 19).

In a preferred aspect, the anti-CD38 antibody drug is felzartamab or a biosimilar of felzartamab.

In some embodiments, the drug neutralizing agent is selected form the group: DTT, an anti- idiotypic antibody specific for an anti-CD38 antibody drug, or CD38-Fc.

In one aspect, the drug neutralizing agent binds to the anti-CD38 antibody drug and blocks and/or displaces said drug from binding to CD38 on red blood cells or platelets.

In one embodiment, the anti-CD38 neutralizing agent is used in molar-excess to the anti-CD38 antibody. In one embodiment, the anti-CD38 neutralizing agent is used in concentration-excess to the anti-CD38 antibody. In one embodiment, the anti-CD38 neutralizing agent is used in molarexcess and/or concentration-excess to the anti-CD38 antibody.

In one aspect, the anti-idiotypic antibody specific for the anti-CD38 antibody (e.g., felzartamab) is used in molar-excess to the anti-CD38 antibody of about 2-fold. In one aspect, the anti-idiotypic antibody specific for the anti-CD38 antibody (e.g., felzartamab) is used in concentration-excess to the anti-CD38 antibody of about 2-fold. In one aspect, the anti-idiotypic antibody specific for the anti-CD38 antibody (e.g., felzartamab) is used in molar-excess and/or concentration-excess to the anti-CD38 antibody of about 2-fold.

In another aspect, the drug neutralizing agent is a CD38-Fc and is used in molar-excess to the anti-CD38 antibody of about 2 to 3-fold. In one aspect, the drug neutralizing agent is a CD38-Fc and is used in concentration-excess to the anti-CD38 antibody of about 2 to 3-fold. In one aspect, the drug neutralizing agent is a CD38-Fc and is used in molar-excess and/or concentrationexcess to the anti-CD38 antibody of about 2 to 3-fold.

In another aspect, the drug neutralizing agent is a CD38-Fc and is used in molar-excess to the anti-CD38 antibody of about 2.5-fold. In another aspect, the drug neutralizing agent is CD38-Fc and is used in concentration-excess to the anti-CD38 antibody of about 2.3-fold. In a preferred aspect, the drug neutralizing agent is an anti-idiotypic antibody specific for an anti- CD38 antibody drug, wherein the anti-idiotypic antibody comprises a variable heavy chain comprising an HCDR1 of the amino acid sequence YSFSNYWIS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 2), an HCDR3 of the amino acid sequence SRGAGMDY (SEQ ID NO: 3), and a variable light chain comprising an LCDR1 of the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 4), an LCDR2 of the amino acid sequence LLIYADNNRPS (SEQ ID NO: 5), an LCDR3 of the amino acid sequence GSYDESSNSM (SEQ ID NO: 6).

In a further aspect the anti-idiotypic antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence

QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence

DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8).

In one aspect, the serological assay is an Indirect Coombs test, also referred to as the indirect antiglobulin test (IAT).

In an additional embodiment, use of an anti-idiotypic antibody specific for MQR202 to neutralize or prevent MQR202 interference in in vitro serological test systems is provided.

In one aspect, the use of an anti-idiotypic antibody specific for MQR202 to neutralize or prevent MQR202 interference in in vitro serological assay systems does not affect red blood cell surface antigens.

In one aspect, the use as enclosed above comprises an anti-idiotypic antibody specific for an anti-CD38 antibody drug, wherein the anti-idiotypic antibody comprises a variable heavy chain (VH) comprising an HCDR1 of the amino acid sequence YSFSNYWIS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 2), an HCDR3 of the amino acid sequence SRGAGMDY (SEQ ID NO: 3), and a variable light chain (VL) comprising an LCDR1 of the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 4), an LCDR2 of the amino acid sequence LLIYADNNRPS (SEQ ID NO: 5), an LCDR3 of the amino acid sequence GSYDESSNSM (SEQ ID NO: 6).

In one aspect, the use as disclosed above comprises an anti-idiotypic antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8).

In one aspect the anti-idiotypic antibody MQR09292 specific for felzartamab is used in molarexcess to felzartamab of about 2-fold. In one aspect, the anti-idiotypic antibody MQR09292 specific for the anti-CD38 antibody felzartamab is used in concentration-excess to felzartamab of about 2-fold. In one aspect, the anti-idiotypic antibody MQR09292 specific for the anti-CD38 antibody felzartamab is used in 2-fold concentration-excess to felzartamab. In one aspect, the anti-idiotypic antibody MQR09292 specific for the anti-CD38 antibody felzartamab is used in molar-excess and/or concentration-excess to felzartamab of about 2-fold.

In one aspect, the use of an anti-idiotypic antibody specific for MQR202 to neutralize or prevent MQR202 interference is in a direct antiglobulin test (DAT) in vitro, or in an Indirect Coombs test, also referred to as the indirect antiglobulin test (IAT).

In one embodiment a kit is provided comprising i) an anti-idiotypic antibody comprising a heavy chain variable domain (VH) comprising the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSKTRYSP SFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVTVSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSGVPDR FSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8) and ii) a panel comprising a set of cells suitable for IAT and NaCI testing.

In one embodiment the RBCs and/or platelets are reagent RBCs and/or reagent platelets, respectively.

In one embodiment the serological assay is an antibody detection (screening) test. In one embodiment the serological assay is an Indirect Coombs Test, also named Indirect Antiglobulin Test (IAT). In one embodiment the serological assay is an antibody identification panel. In one embodiment the serological assay is a direct antiglobulin tests (DAT). In one embodiment the serological assay is an anti-human globulin (AHG) crossmatch. Working Examples

Example 1 : Antibody screening test

Stage 1 Antibody identification in the BioRad system

Identification of commercially available plasma samples to be used for further testing (i.e. in stages 2 and 3). Six human plasma samples from drug naive healthy volunteers were ordered at Sera Laboratories International Ltd. (Cat. # HMPLEDTA3) (Lot# BRH1221754, BRH1221755, BRH1221756, BRH1221757, BRH1221758, BRH1221759) Method:

Column agglutination technique, ID-DiaPanel (#45161 , BioRad) Coombs Anti-IgG (#50540, BioRad)

Results:

Negative antibody screening test for all six plasma samples (2 samples shown in Figure 1). Selection of plasma sample L#BRH1221755, -56 and -58 for further testing (i.e. in stages 2 and 3).

Stage 2_1 : Antibody identification test at six different concentrations of MOR202

Testing the influence of different concentrations of MOR202 on the antibody identification test. Three human plasma samples selected in stage 1 were spiked with the following concentrations of MOR202: 0 pg/mL, 3 pg/mL, 10 pg/mL, 30 pg/mL, 100 pg/mL, 300 pg/mL.

Method:

Column agglutination technique, ID-DiaPanel (#45161 , BioRad) Coombs Anti-IgG (#50540, BioRad). Plasma samples L#BRH1221755, -56 und -58, MOR202 stock solution 10.2 pg/mL (Lot # 160915_125SLR04*2).

Results:

MOR202 induces positive reactions (agglutinations) in the antibody identification test for all tested human plasma samples. Agglutination already visible at a drug concentration of 3 pg/mL. No reactivity was observed at 0 pg/mL MOR202. Reactions are in general very weak in comparison to other CD38- targeting antibodies as reported by Oostendorp et al., 2015. Strongest reaction visible at a MOR202 concentration of 10 pg/mL. In a few cases, the reaction strength decreases with increasing concentration of MOR202 (Figure 2).

Stage 2_2: Antibody identification in the Grifols system Confirmation of the influence of MOR202 in a further immunohematologic testing system (DG Gel Cards, Grifols). MOR202 was tested at three concentrations: 10 pg/mL, 30 pg/mL, 100 pg/mL. The same three plasma samples selected in stage 1 and analyzed in stage 2_1 were used in this antibody identification test system.

Method:

Column agglutination technique (DG Gel Cards, Grifols) Material: Plasma samples L#BRH1221755, -56 und -58. MOR202 stock solution 10.2 pg/mL (Lot # 160915_125SLR04*2)

Results:

Antibody identification test: overall stronger agglutination visible (Figure 3) compared to Biorad system. No concentration dependency of the reaction strengths (not shown).

Stage 3_1 : Inhibition of interference induced by MOR202 on blood compatibility testing at 300 pg/mL by using DTT, MOR09292 or CD38-Fc

Objective:

Confirmation of MOR202 specificity on assay interference and testing the neutralization of MOR202 induced agglutination using Dithiothreitol (DTT), the anti-idiotypic antibody MOR09292 or CD38-Fc. MOR202 was tested at a concentration of 300 pg/mL and three individual plasma samples were used. A representative allo-antibody control was included to prove the integrity of the Duffy-antigen (Fya).

Material: Plasma samples L#BRH1221755, -56 und -58

DTT (SIGMA-Aldrich), 0.2 M in PBS

BioRad Q.C.

Sample 2 containing alloantibody anti-Fya (BioRad)

MOR202 stock solution: 20.2 pg/mL

MOR09292 stock solution: 19.1 pg/mL

Used in molar-excess to MOR202: ~ 2-fold

Concentration-excess to MOR202: 2-fold

CD38-Fc stock solution : 20.0 pg/mL

Used in molar-excess to MOR202: 2.5-fold

Concentration-excess to MOR202: ~ 2.3-fold

Methods: Antibody identification test: Column agglutination technique (BioRad)

Table 1: Experimental Set-up Stage 3_1

Results: Agglutination triggered by MOR202 was inhibited by DTT (Figure 4), MOR09292 (Figure 7) and CD38-Fc (not shown). A denaturing influence of the neutralizing reagents on the Duffy-antigen could be excluded by the allo-antibody control (Figures 8 and 9).

Claims

1. A method to reduce or abolish interference of an anti-CD38 antibody comprising a human antibody Fc region in a serological assay, wherein the method comprises the following steps:

(a) adding an anti-CD38 antibody-neutralizing agent to a serum or plasma sample from a subject who has received treatment with said anti-CD38 antibody comprising a human antibody Fc region; and

(b) performing said serological assay of the plasma sample after step (a), wherein said serological assay utilizes red blood cells and/or platelets.

2. The method according to claim 1, wherein the anti-CD38 antibody comprises an HCDR1 of the amino acid sequence SYYMN (SEQ ID NO: 12), an HCDR2 of the amino acid sequence GISGDPSNTYYADSVKG (SEQ ID NO: 13), an HCDR3 of the amino acid sequence DLPLVYTGFAY (SEQ ID NO: 14) an LCDR1 of the amino acid sequence: SGDNLRHYYVY (SEQ ID NO: 15), an LCDR2 of the amino acid sequence: GDSKRPS (SEQ ID NO: 16), and an LCDR3 of the amino acid sequence: QTYTGGASL (SEQ ID NO: 17).

3. The method according to claim 1 or claim 2, wherein the anti-CD38 antibody comprises a heavy chain variable domain (VH) of the amino acid sequence: QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPS NTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGT LVTVSS (SEQ ID NO: 18) and a light chain variable domain (VL) of the amino acid sequence: DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIP ERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ (SEQ ID NO: 19).

4. The method according to any of the preceding claims, wherein the anti-CD38 antibody is felzartamab or a biosimilar of felzartamab. The method according to any of the preceding claims, wherein the anti-CD38 neutralizing agent is used in molar-excess and/or concentration-excess to the anti-CD38 antibody. The method according to any of the preceding claims, wherein the anti-CD38 antibodyneutralizing agent is selected from DTT, an anti-idiotypic antibody specific for the anti- CD38 antibody, and CD38-Fc. The method according to claim 6, wherein the anti-CD38 antibody-neutralizing agent binds to and/or interacts with the anti-CD38 antibody and blocks and/or displaces said anti-CD38 antibody from binding to CD38 on red blood cells or platelets. The method according to claim 6, or claim 7, wherein the anti-CD38 antibodyneutralizing agent is an anti-idiotypic antibody specific for an anti-CD38 antibody, wherein the anti-idiotypic antibody comprises a variable heavy chain comprising an HCDR1 of the amino acid sequence YSFSNYWIS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 2), an HCDR3 of the amino acid sequence SRGAGMDY (SEQ ID NO: 3), and a variable light chain comprising an LCDR1 of the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 4), an LCDR2 of the amino acid sequence LLIYADNNRPS (SEQ ID NO: 5), an LCDR3 of the amino acid sequence GSYDESSNSM (SEQ ID NO: 6). The method according to claim 8, wherein the anti-idiotypic antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSK TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVT VSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSG VPDRFSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8). The method according to any one of claims 6 to 9, wherein the anti-idiotypic antibody is used in molar-excess and/or concentration-excess to the anti-CD38 antibody of about 2- fold. The method according to any of the preceding claims, wherein the serological assay is an Indirect Coombs test. Use of an anti-idiotypic antibody specific for felzartamab to neutralize or prevent interference of felzartamab in a serological assay. The use according to claim 12, wherein the anti-idiotypic antibody does not affect red blood cell surface antigens. The use according to claim 12 or claim 13, wherein the anti-idiotypic antibody is specific for an anti-CD38 antibody, and wherein the anti-idiotypic antibody comprises a variable heavy chain comprising an HCDR1 of the amino acid sequence YSFSNYWIS (SEQ ID NO: 1), an HCDR2 of the amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 2), an HCDR3 of the amino acid sequence SRGAGMDY (SEQ ID NO: 3), and a variable light chain comprising an LCDR1 of the amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 4), an LCDR2 of the amino acid sequence LLIYADNNRPS (SEQ ID NO: 5), an LCDR3 of the amino acid sequence GSYDESSNSM (SEQ ID NO: 6). The use according to claim 14, wherein the anti-idiotypic antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSK TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVT VSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSG VPDRFSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID NO: 8). The use according to any of claims 12 to 15, wherein the serological assay is an Indirect Coombs test. The use according to any of claims 12 to 16, wherein the anti-idiotypic antibody is used in molar-excess and/or concentration-excess to the anti-CD38 antibody of about 2-fold. A kit comprising i) an anti-idiotypic antibody comprises a heavy chain variable domain (VH) comprising the amino acid sequence QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWISWVRQMPGKGLEWMGIIDPASSK

TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARSRGAGMDYWGQGTLVT VSS (SEQ ID NO: 7) and a light chain variable domain (VL) comprising the amino acid sequence DIVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYADNNRPSG VPDRFSGSKSGTSASLAITGLQSEDEADYYCGSYDESSNSMVFGGGTKLTVL (SEQ ID

NO: 8), and ii) red blood cells and/or platelets suitable for IAT and NaCI testing.