WO2023186905A1

WO2023186905A1 - A method of treating a hematological cancer following screening for cd1d positive tumor cells

Info

Publication number: WO2023186905A1
Application number: PCT/EP2023/058016
Authority: WO
Inventors: Jurjen Matthijs RUBEN; Roeland Lameris; Johannes Jelle VAN DER VLIET; Thilo Alexander RIEDL; Anton Egbert Peter Adang; Paul Willem Henri Ida PARREN
Original assignee: LAVA Therapeutics N.V.
Priority date: 2022-03-29
Filing date: 2023-03-28
Publication date: 2023-10-05

Abstract

Disclosed herein is a method of treating a hematological cancer in a subject, such as a subject diagnosed with CLL, MM or AML, the method comprising screening the subject by analyzing cells in a biological sample obtained from the subject, and if the cells exhibit a CD1d Fluorescence Index of at least 1.1 (e.g. at least 1.5) the subject is treated with a bispecific antibody that comprises a binding moiety that binds CD1d and a binding moiety that binds a Vγ9Vδ2-T cell receptor, such as the Vγ9Vδ2-T cell engaging bispecific antibody 1D12-5C8var.

Description

A METHOD OF TREATING A HEMATOLOGICAL CANCER FOLLOWING SCREENING FOR CD1D POSITIVE TUMOR CELLS

BACKGROUND

[0001] CD Id is a member of the CD1 (cluster of differentiation 1) family of glycoproteins (including CD la, CD lb, CDlc, CD Id and CDle) and is expressed on the surface of various human cells, including antigen presenting cells (APC). CD Id is structurally related to the major histocompatibility complex (MHC) class I glycoprotein and is involved in the presentation of (glyco)lipid antigens to CDld restricted T cells, including invariant (i)NKT cells, also termed type 1 NKT cells. Based on the applicant’s analyses of patient samples, as well as literature reports, it is expected that CDld will be expressed on tumor cells by the majority of patients with chronic lymphocytic leukemia (CLL), multiple myeloma (MM). In acute myeloid leukemia (AML) expression is particularly observed on myelomonocytic/monocytic subtypes (formerly diagnosed as FAB M4/M5), which makes it a potential target for the treatment of these disorders.

[0002] Antibody lD12-5C8var is a Vy9V52-T and iNKT cell engaging bispecific antibody of 27.3 kDa that engages Vy9V52-T cells and iNKT cells in the killing of tumor cells in a tumor targetdependent manner. Vy9V52-T cells form a relatively homogeneous T cell subset (approximately 1- 10% of all cluster of differentiation (CD)3+ T cells) that are activated via their interaction with phosphoantigen bound butyrophilin (BTN)3 Al and BTN2A1 molecules to induce cell death in a wide range of malignant cells in a human leukocyte antigen (HL A) -independent fashion. Antibody 1D12- 5C8var consists of two VHH (variable domain of a heavy chain-only antibody) domain antibodies linked via a 5 -amino acid glycine-serine linker. One VHH recognizes the V52 chain of the y5-T cell receptor (TCR), the other arm is specific for the tumor antigen CDld. Antibody lD12-5C8var thereby targets Vy9V52-T cells to tumor cells and induces their potent and specific killing. The latter interaction also causes the activation of a second effector cell type: iNKT cells. The unique features of antibody lD12-5C8var and the capabilities of the unique effector cells that it activates have the potential of making a substantial beneficial impact in the treatment of patients with CD Id-expressing CLL, MM, and AML and potentially other CDld expressing malignancies including T-ALL, colorectal cancer, lung cancer, head & neck cancer, breast cancer, renal cancer, melanoma and neuroblastoma. [0003] . Despite current treatment options, there is still an unmet need for patients diagnosed with these diseases as the vast majority of patients will experience relapse of disease, become refractory to, or develop resistance to existing therapies, and will eventually succumb to the consequences of the disease.

[0004] Antibody lD12-5C8var is disclosed in PCT Published Patent Application No. 2020060405 as SEQ ID NO:87 (SEQ ID NO:1 herein). WO 2020060405 also discloses the preparation of antibody lD12-5C8var and related antibodies, methods of formulating pharmaceutical compositions comprising antibody lD12-5C8var, methods of treating diseases, including CLL, MM, and AML, with antibody lD12-5C8var and related antibodies, and dosing and dose regimens using pharmaceutical compositions that comprise antibody lD12-5C8var. WO 2020060405 and related application WO 2020060406 are herein incorporated by reference in its entirety for all purposes.

[0005] The applicant has now found that Vy9V52-T and iNKT-cell mediated tumor cell lysis occurs in CLL-, MM-, or AML-presenting patient samples displaying a CDld Fluorescence Index, e.g. a Molecules of Equivalent Soluble Fluorochrome (MESF) index, of > 1.1. Vy9V52-T cell mediated tumor cell lysis is consistently observed in patient samples displaying a CDld MESF index of > 1.5 (see Figure 1). Using a MESF index allows the robust assessment of the CDld expression level in patient samples independent of flow cytometer settings, and in a cross-platform manner. Therefore, the present disclosure describes a method of treating a hematological cancer in a subject diagnosed with for examples CLL, MM or AML or other CD 1 d-positive hematological cancers. The method comprises screening the subject by labeling cells in a biological sample obtained from the subject, analyzing the labeled cells using flow cytometry, and if the subject’s cells exhibit a calculated CDld MESF Index of at least 1.1 (e.g. at least 1.1, 1.2, 1.3, 1.4 or especially 1.5) the subject is treated with a bispecific antibody that comprises a binding moiety that binds CDld and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var. Alternatively, the present disclosure a method comprising screening the subject by labeling tumor cells, e.g. obtained from a needle biopsy in which the cells are dissociated, analyzing the labeled dissociated cells using flow cytometry, and if the subject’s dissociated cells exhibit a calculated CDld MESF Index of at least 1.1 (e.g. at least 1.1, 1.2, 1.3, 1.4 or especially 1.5) the subject is treated with a bispecific antibody that comprises a binding moiety that binds CDld and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var It is anticipated that patients with CDld-positive malignancies will be eligible for treatment with a bispecific antibody that comprises a binding moiety that binds CDld and a binding moiety that binds a Vy9V52-T cell receptor, such as antibody lD12-5C8var if their calculated CDld MESF index is >1.5. SUMMARY

[0006] Disclosed herein is a method of treating a subject with a hematological cancer, the method comprising:

I. a prescreening step comprising: i. providing a biological cell sample comprising mononuclear cells, wherein said sample is obtained from a subject with a hematological cancer; ii. labeling a first portion of cells via (a) an antibody that binds CD Id and (b) one or more antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with said hematological cancer; iii. labeling a second portion of cells via (a) an isotype control antibody and (b) the same antibodies as in ii. (b) above; iv. analyzing the labelled cells using flow cytometry; v. selecting the flow cytometry data set corresponding to tumor cells, and vi. calculating a CD Id Fluorescence Index from the acquired fluorescence for tumor cells labeled via said antibody that binds CD Id and the acquired fluorescence for tumor cells labeled via said isotype control antibody; and

II. a treatment step wherein a prescreened subject whose sample exhibits a CD Id Fluorescence Index of at least 1.1 (e.g. at least 1.5) is treated with a bispecific antibody that comprises a binding moiety that binds CD Id and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var.

[0007] In some cases, the hematological cancer is selected from chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and multiple myeloma (MM).

[0008] In some cases, the biological sample is a whole blood sample or a bone marrow sample, and mononuclear cells are isolated using a ficoll gradient, followed by the removal of thrombocytes and lysis of erythrocytes, and subsequently washing with an excess of PBS. In some cases, the biological sample is a whole blood sample or a bone marrow sample, and erythrocytes are lysed after which the cell sample is washed with an excess of PBS. In some cases, the subject is diagnosed with CLL, MM or AML, and the cell sample is taken from the subject and a first plurality of cells therein are analyzed. In some cases, the subject is diagnosed with CLL, MM or AML, and a bone marrow sample is taken from the subject and a first plurality of cells therein are analyzed. [0009] In some cases, the cells are primarily peripheral blood mononuclear cells (PBMCs). In some cases, the cells consist essentially of PBMCs. In some cases, the cells consist of PBMCs.

[0010] In some cases, the cells are primarily bone marrow mononuclear cells (BMMCs). In some cases, the cells consist essentially of BMMCs. In some cases, the cells consist of BMMCs.

[0011] In some cases, the sample is a lymph node sample, from which the cell sample can, e.g., be acquired and processed as described in Vuylsteke et al. (2004) Cancer Res 64:8456. In some cases, the sample is a (fine needle) biopsy, from which the cell sample can, e.g., be acquired and process as described in Gill et al. (2019) Gut 68: 1493.

[0012] In some cases, the method further comprises dividing the cell sample into a first plurality of cells and a second, approximately equal, plurality of cells and suitably labeling the cells.

[0013] In some cases, the method comprises isolating a second cell sample from the subject.

[0014] In some cases, the first portion of cells is labelled with (a) an antibody that binds CDld and (b) antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells of subjects with CLL. In some cases, the second portion of cells is labelled with

(a) an isotype control antibody and (b) the same antibodies that are used to label the first portion of cells.

[0015] In some cases, the first portion of cells is labelled with (a) an antibody that binds CDld and

(b) antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells of subjects with AML. In some cases, the second portion of ells is labelled with

[0016] In some cases, the first portion of cells is labelled with (a) an antibody that binds CDld and

(b) antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells of subjects with MM. In some cases, the second portion of cells is labelled with (a) an isotype control antibody and (b) the same antibodies that are used to label the first portion of cells.

[0017] As defined herein, an “isotype control antibody” is a negative control to help determine the contribution of non-specific staining. The isotype control antibody has the same isotype as the antibody that binds CD 1 d, but lacks specificity to CD 1 d. The antibody that binds CD 1 d and the isotype control antibody are typically labelled with the same fluorochrome, or they are both detected using a secondary antibody labelled with a fluorochrome.

In some cases, the subject is diagnosed with CLL, and the antibodies defined in (b) above bind to at least one target selected from group consisting of CD5, CD19, CD23, CD43, CD45 and CD79b. Thus, in some cases, the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with CLL specifically bind to CD5, CD19, CD23, CD43, CD45 and/or CD79b. Optionally, the antibodies defined in (b) further include antibodies that bind CD20.

[0018] In some cases, the subject is diagnosed with CLL, and the antibodies defined in (b) above that bind to multiple known antigens comprise antibodies that specifically bind to multiple targets including CD5, CD23 and CD43. In some cases, the subject is diagnosed with CLL, and the antibodies defined in (b) above that bind to multiple known antigens comprise antibodies that specifically bind to multiple targets including CD5, CD19, CD23, CD43, CD45 and CD79b.

[0019] In some cases, the subject is diagnosed with MM, and the antibodies defined in (b) above bind to at least one target selected from group consisting of CD19, CD38, CD45, CD56 and CD138. Thus, in some cases, the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with MM specifically bind to CD19, CD38, CD45, CD56 and/or CD138.

[0020] In some cases, the subject is diagnosed with MM, and the antibodies defined in (b) above that bind to one or more known antigens comprise antibodies that specifically bind to at least one target selected from group consisting of CD38, CD56 and CD138. In some cases, the subject is diagnosed with MM, and the antibodies defined in (b) above that bind to one or more known antigens comprise antibodies that specifically bind to at least one target selected from group consisting of CD 19, CD38, CD45, CD 56 and CD138.

[0021] I In some cases, the subject is diagnosed with AML, and the antibodies defined in (b) above bind to at least one target selected from group consisting of CD7, CD 13, CD 19, CD22, CD33, CD34, CD45, CD117 and CD133. Thus, in some cases, the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with AML specifically bind to CD7, CD13, CD19, CD22, CD33, CD34, CD45, CD117 and/or CD133.

[0022] In some cases, the subject is diagnosed with AML, and the antibodies defined in (b) above that bind to one or more known antigens comprise antibodies that specifically bind to at least one target selected from group consisting of CD7, CD13, CD22, CD33, CD34, CD117 and CD133. In some cases, the subject is diagnosed with AML, and the antibodies defined in (b) above that bind to one or more known antigens comprise antibodies that specifically bind to at least one target selected from group consisting of CD7, CD13, CD19, CD22, CD33, CD34, CD45, CD117 and CD133.

[0023] In some cases, the analysis of the cell sample further includes staining for cell viability with a dye, such as 7-AAD. [0024] In some cases, the aforementioned antibodies that bind to one or more known antigens that are expressed on tumor cells or known to be absent on tumor cells in subjects with hematological cancer (e.g. CLL, MM or AML) are also labeled by conjugation to one or more fluorochromes. Preferably, the fluorochromes that conjugate to antibodies that bind to one or more known antigens that are expressed in subjects with a hematological cancer are different from the fluorochrome that conjugates to the antibody that specifically bind to CD Id and the isotype control antibody.

[0025] In some cases, the antibodies are directly conjugated to a fluorochrome.

[0026] In some cases, the antibodies are detected via a secondary antibody which carries the fluorochrome. In one embodiment, antibodies are bound or targeted by a secondary antibody conjugated to a fluorochrome. In one embodiment, such a secondary antibody targets the constant region of the CD Id binding- and isotype control antibody.

[0027] After labeling with the antibodies, the cells are washed and analyzed with a flow cytometer.

[0028] Subsequently, on the basis of the obtained flow cytometry data set, a data set corresponding to the tumor cells is being selected on the basis of the target expression signal generated by the antibodies specified in (b). In some cases, this is performed by gating on the viable cell population in the FSC/SSC (forward scatter / side scatter) and subsequently gating the tumor cells on the basis of the corresponding target expression signal generated by the antibodies specified in (b).

[0029] The data set corresponding to the tumor cells can then be used to calculate the CD Id Fluorescence Index by dividing the signal generated by the CDld binding antibody on the selected tumor cell population with the signal generated by the isotype control antibody.

[0030] In some cases, detecting the CDld expression level comprises detecting the Molecules of Equivalent Soluble Fluorochrome (MESF) on tumor cells, in a cell sample labelled with a fluorochrome conjugated to an antibody that binds CDld (“MESF-1”). In some cases, detecting the CDld expression level further comprises detecting the MESF on tumor cells, in a cell sample labelled with the same fluorochrome conjugated to an isotype control antibody (“MESF-2”). In some cases, the fluorochrome is Brilliant Violet 421 (BV421). The MESF is detected and measured by flow cytometry. In some cases, the method further comprises generating a CDld Molecules of Equivalent Soluble Fluorochrome (MESF) Index by dividing MESF-1 by MESF-2. Thus, in some cases, the CDld MESF index is calculated according to the formula: CDld MESF index = CDld MESF / Isotype MESF.

[0031] In some cases, a subject diagnosed with CLL, having a CDld Fluorescence Index, such as a MESF Index, of at least 1.1 (e.g. at least 1.5) according to the prescreening step hereinabove, is treated with antibody lD12-5C8var. [0032] In some cases, a subject diagnosed with AML, having a CD Id Fluorescence Index, such as a MESF Index, of at least 1.1 (e.g. at least 1.5) according to the prescreening step hereinabove, is treated with antibody lD12-5C8var.

[0033] In some cases, a subject diagnosed with MM, having a CD Id Fluorescence Index, such as a MESF Index, of at least 1.1 (e.g. at least 1.5) according to the prescreening step hereinabove, is treated with antibody lD12-5C8var.

[0034] In one aspect, the subject exhibits a CD Id Fluorescence Index, such as a MESF Index, of at least 1,1, such as at least 1,2, for example at least 1.3, such as at least 1.4, for example at least 1.5, such as at least, 1.6, for example at least 1.7, such as at least 1.8, for example at least 1.9, such as at least 2, for example at least 3, such as at least 4, for example at least 5, such as at least 6, for example at least 7, such as at least 8, for example at least 9, such as at least 10, for example at least 12, such as at least 15, for example at least 20, or from 1.5 to 128, e.g. from 2 to 128.

[0035] The prescreened subject selected for therapy may be administered a bispecific antibody that comprises a binding moiety that binds CD Id and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var or a variant of antibody lD12-5C8var by any suitable route, but will typically be parenteral, such as intravenous, intramuscular or subcutaneous as a bolus or gradually by fractionated or continuous infusion. Effective dosages and the dosage regimens depend on the disease or condition to be treated and may be determined by the persons skilled in the art. Conveniently, the antibody may be presented as a solution for intravenous (IV) infusion, and may be a sterile, colorless, clear liquid or may be presented as a solution for subcutaneous (SC) infusion and may be a sterile, colorless, clear liquid.

[0036] The antibody may also be administered as part of a combination therapy, i.e., combined with other therapeutic agents relevant for the disease or condition to be treated. Accordingly, in one embodiment, antibody lD12-5C8var is administered in combination with one or more further therapeutic agents, such as cytotoxic, chemotherapeutic or anti-angiogenic agents, or other immunotherapeutic treatment including check point blockers or check point agonists or cell therapy treatments including adoptive transfer of (expanded) autologous or allogeneic cells (T cells, gammadelta T cells, NK cells or iNKT cells) or engineered cells including CAR-Ts. Such combined administration may be simultaneous, separate or sequential. In a particular embodiment, the present disclosure provides a method of treating a hematological cancer, such as CLL, AML or MM, comprising selecting a subject for treatment using the prescreening step above, followed by administration to the subject in need thereof antibody lD12-5C8var in combination with one or more therapeutic agents, radiotherapy and/or surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGURE 1 provides a flow cytometry data example for CLL. PBMC of a patient diagnosed with a CLL were labeled with antibodies targeting CD19 and CD5. The CLL cells residing in the lymphocyte population was selected based on the FSC/SSC characteristics. From this gate, the CLL cells were selected based on the co-expression of CD19 and CD5.

[0038] FIGURE 2 shows a correlation between CD Id MESF index (x-axis) and specific CLL, MM or AML tumor cell lysis assessed in vitro (y-axis) following Vy9V52-T cell engagement with antibody lD12-5C8var. The thin dotted lines represent MF index values of 1.5 (left) and 2.0 (right). Vy9V52- T cells, expanded from healthy donors, mediated tumor cell lysis of CLL cells in patient peripheral blood mononuclear cell samples, and of MM-, or AML cells in patient bone marrow mononuclear cells cultured in the presence of 10 - 50nM of antibody lD12-5C8var.

DETAILED DESCRIPTION

Overview

[0039] Disclosed herein are methods for detecting the CD Id expression level in cells isolated from patients diagnosed with a hematological cancer, as well as methods for screening patients diagnosed with a hematological cancer for CD Id-positive cells, together with a treatment step, wherein patients diagnosed with a hematological cancer and having a certain CD Id expression level in mononuclear cells (e.g. PBMCs or BMMCs) are treated with a CD Id-binding antibody, such as antibody 1D12- 5C8var.

Definitions

[0040] The term “antibody” as used herein is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a binding half-life, for example to a tumor cell and/or an effector cell, of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The binding region (or binding domain) which interacts with an antigen, comprises variable regions of both the heavy and light chains of the immunoglobulin molecule or a binding domain of a heavy chain for a VHH or heavy chain-only antibody. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. In some embodiments, however, the Fc region of the antibody has been modified to become inert, “inert” means an Fc region which is at least not able to bind any Fey Receptors, induce Fc-mediated cross-linking of FcRs, or induce FcR-mediated cross-linking of target antigens via two Fc regions of individual proteins, such as antibodies. In a further embodiment, the inert Fc region is in addition not able to bind Clq. In one embodiment, the antibody contains mutations at positions 234 and 235 (Canfield and Morrison (1991) J Exp Med 173:1483), e.g. a Leu to Phe mutation at position 234 and a Leu to Glu mutation at position 235. In another embodiment, the antibody contains a Leu to Ala mutation at position 234, a Leu to Ala mutation at position 236 and a Pro to Gly mutation at position 329.

[0041] As indicated above, the term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically interact, such as bind, to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include (i) a Fab’ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains, or a monovalent antibody as described in W02007059782; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CHI domains; and (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody; and (v) a VHH fragment consisting of the VH domain of a heavy chain-only antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present disclosure, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present disclosure are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, bispecific and multispecific antibody, antibody-drug conjugates, engineered antibodies and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype or maybe a fragment without an isotype.

[0042] The term “isotype” as used herein, refers to the immunoglobulin (sub)class (for instance IgGl, IgG2, IgG3, IgGl, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgGlm(za) and IgGlm(f) that is encoded by heavy chain constant region genes. Thus, in one embodiment, the antibody comprises a heavy chain of an immunoglobulin of the IgGl class or any allotype thereof. Further, each heavy chain isotype can be combined with either a kappa (K) or lambda (A) light chain.

[0043] The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR). Humanization of a VHH antibody fragment can be achieved by grafting of the three non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR), or alternatively by identifying framework residues that differ between the (closest) human germ line gene and the non-human antibody and substituting individual or combinations of amino acid positions that differ from the human germ line sequence in the non-human antibody. For the former method, in order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. The amino acid sequence of an antibody of non-human origin is distinct from antibodies of human origin, and therefore a non-human antibody is potentially immunogenic when administered to human patients. However, despite the non-human origin of the antibody, its CDR segments are responsible for the ability of the antibody to bind to its target antigen and humanization aims to maintain the specificity and binding affinity of the antibody. Thus, humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity risk in man while such humanized antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.

[0044] The terms "binds” or “specifically binds" as used herein in the context of an antibody, refers to the binding of a binding moiety or binding molecule to a predetermined antigen or target (e.g. human CD Id or human Vdelta2) to which binding typically is with an affinity corresponding to a KD of about IO"⁶ M or less, e.g. 10'⁷ M or less, such as about 10'⁸ M or less, such as about ICT⁹ M or less, about 10’ ¹⁰ M or less, or about IO ¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the binding moiety or binding molecule as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely- related antigen. The degree with which the affinity is lower is dependent on the KD of the binding moiety or binding molecule, so that when the KD of the binding moiety or binding molecule is very low (that is, the binding moiety or binding molecule is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular interaction between the antigen and the binding moiety or binding molecule. Alternatively, the (apparent) binding affinity or KD is assessed by binding of the antibody to cells expressing the target and by determining the half-maximal binding level using flow cytometric techniques.

[0045] “SEQ ID NO:1” herein refers to the following sequence:

[0046] EVQLVESGGGLVQAGGSLRLSCAASGSMFSDNVMGWYRQAPGKQRELVATIRTGG STNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCRHTIPVPSTPYDYWGQGTQV TVSSGGGGSEVQLLESGGGSVQPGGSLRLSCAASGRPFSNYAMSWFRQAPGKEREFVSAIS WSGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAAQFSGADYGFGRLGIR GYEYD YWGQGTQ VTVS S .

[0047] The term “a variant of antibody lD12-5C8var” includes any antibody that comprises the same 6 CDR sequences as antibody lD12-5C8var or a variant of said CDR sequences wherein each CDR contains one, two, three, four or five amino acid modifications (substitutions, deletions or additions) relative to the corresponding CDR sequence of antibody lD12-5C8var. The 6 CDR sequence of antibody lD12-5C8var are the following: NYAMS (5C8var CDR1 - SEQ ID NO:2); AISWSGGSTSYADSVKG (5C8var CDR2 - SEQ ID N0:3); QFSGADYGFGRLGIRGYEYDY (5C8var CDR3 - SEQ ID N0:4): DNVMG (1D12 CDR1 - SEQ ID N0:5); TIRTGGSTNYADSVKG (1D12 CDR2 - SEQ ID N0:6); TIPVPSTPYDY (1D12 CDR3 - SEQ ID N0:7). Variants of antibody lD12-5C8var also include variants wherein 5C8var and 1D12 are not linked via a peptide linker, but for example via Fc sequences attached to the VHH domains.

Chronic Lymphocytic Leukemia

[0048] Chronic Lymphocytic Leukemia (CLL) is a common leukemia in Western countries, with an incidence of approximately 4.7 cases per 100,000 people in the USA. In Western Europe the incidence of CLL has been increasing during the past years reaching an incidence of up to 5.27 per 100,000 in the UK. The disease has a male predominance and a median age of diagnosis of ~ 70 years. CLL is caused by the monoclonal expansion of mature-appearing functionally incompetent neoplastic B lymphocytes and the progressive accumulation of these cells in blood, secondary lymphatic tissues, and bone marrow. When used herein, the term “CLL” refers to B-CLL, unless specified otherwise. CLL has a highly variable presentation and clinical course. The majority of patients with CLL are initially asymptomatic, have indolent disease, and are managed with a watch-and-wait approach. In time, the majority of patients will require treatment. There is no single agreed-upon standard frontline treatment regimen for all symptomatic CLL, mostly due to differences in patient age and fitness. There are several initial treatment options and most have not been directly compared. While overall survival rates with the different available regimens are similar, they differ in their rates of complete remission, time to progression, and associated toxicities. Even in case of excellent responses to first line therapy, most patients will relapse with the need for subsequent therapy. Relapsed or refractory CLL remains incurable, despite advances in treatment over the last years. When disease progression occurs, especially after treatment with DNA-damaging agents, CLL cells serially accumulate adverse biological features and increasingly develop resistance to therapies. Therefore, novel and more effective therapeutic approaches with alternative mechanisms of action and an acceptable side-effect profile are needed.

[0049] Chronic Lymphocytic Leukemia (CLL) is a type of cancer in which the bone marrow makes too many lymphocytes (a type of white blood cell). CLL can progress slowly affecting older adults, and may not cause any symptoms for years. When symptoms do occur, they may include swollen lymph nodes, fatigue, and easy bruising.

[0050] The guidelines for diagnosis, treatment and response assessment of CLL is described in Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dbhner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood. 2018 21;131(25):2745-60, which is entirely incorporated by reference.

[0051] The diagnosis of CLL can be made through blood tests, which require the presence of >5 x 10⁹/L B lymphocytes in the peripheral blood, sustained for at least 3 months. The clonality of these B lymphocytes can be confirmed by demonstrating immunoglobulin light chain restriction using flow cytometry. The leukemia cells found in the blood smear can be characteristically small, mature lymphocytes with a narrow border of cytoplasm and a dense nucleus lacking discernable nucleoli and partially aggregated chromatin. Gumprecht nuclear shadows, or smudge cells, found as cellular debris, can be additional morphologic features commonly associated with CLL. A small percentage of larger or atypical cells or prolymphocytes can be found admixed with morphologically typical CLL cells. A significant proportion of circulating prolymphocytes (>10%) can indicate a more aggressive form of CLL (with NOTCH1 or genetic TP53 aberrations).

[0052] CLL cells can co-express the surface antigen CD5 together with the B-cell antigens CD19, CD20, and CD23. The levels of surface immunoglobulin, CD20, and CD79b can characteristically low compared with those found on normal B cells. Each clone of leukemia cells can be restricted to expression of either K or X immunoglobulin light chains. The expression of CD5 can also be observed in other lymphocytic malignancies, however, such as mantle cell lymphoma. In some cases, a panel of CD19, CD5, CD20, CD23, K, and X can be used to establish the diagnosis of CLL. In some cases, markers such as CD43, CD79b, CD81, CD200, CD 10, or R0R1 may help to refine the diagnosis.

[0053] In some cases, interphase fluorescence in situ hybridization (FISH) can be performed with peripheral blood lymphocytes to identify cytogenetic lesions in CLL cases. The most common deletions can be in the long arm of chromosome 13 (del(13q)). Additional, frequent chromosomal aberrations can comprise trisomy of chromosome 12 and deletions in the long arm of chromosomes 11 (del(l 1 q)) and in the short arm of chromosome 17 (del(l 7p)). Appropriate stimulation of CLL cells in vitro can enable the performance of conventional karyotyping with enhanced reliability. With this methodology, additional chromosomal aberrations of potential prognostic significance can be identified. Moreover, stimulated metaphase karyotyping can show that leukemia cells with a complex karyotype (i.e., >3 chromosomal abnormalities) may have adverse prognostic significance.

Multiple Myeloma

[0054] Multiple myeloma (MM) is a clonal plasma cell disorder that accounts for approximately 10% of hematological malignancies with an estimated incidence of 4.5-6 per 100,000 people per year in Europe and primarily affects elderly patients with a median age at diagnosis of 72 years. MM is characterized by the neoplastic proliferation of plasma cells producing a monoclonal immunoglobulin (M-protein). The plasma cells proliferate in the bone marrow and often result in extensive skeletal destruction with osteolytic lesions, osteopenia, and/or pathologic fractures. Most patients with MM present with signs or symptoms related to the infiltration of plasma cells into the bone or other organs or to kidney damage from excess light chains. Though the treatment landscape for MM has evolved considerably it remains an incurable disease with relapse of MM considered inevitable. MM is a heterogeneous disease and requires an individualized approach when making treatment decisions taking patient-related factors (e.g. age, comorbidities), disease-related factors (e.g. cytogenetics, disease burden, aggressiveness) and efficacy and toxicity of previous therapies into consideration. There are several treatment options for patients with relapsed or refractory MM with most patients experiencing serial relapses. After each relapse, the disease becomes more aggressive with shortened subsequent progression-free survival. There is therefore a need to develop more effective therapeutic approaches with an acceptable side-effect profile.

[0055] MM, also known as plasma cell myeloma and simply myeloma, is a cancer of plasma cells, a type of white blood cell that normally produces antibodies. Often, no symptoms are noticed initially. As it progresses, bone pain, anemia, kidney dysfunction, and infections may occur. Complications may include amyloidosis.

[0056] Risk factors for multiple myeloma include obesity, radiation exposure, family history, and certain chemicals. Multiple myeloma may develop from monoclonal gammopathy of undetermined significance that progresses to smoldering myeloma. The abnormal plasma cells produce abnormal antibodies, which can cause kidney problems and, occasionally, overly thick blood. The plasma cells can also form a mass in the bone marrow or soft tissue. When one tumor is present, it is called a plasmacytoma; more than one is called multiple myeloma.

[0057] Multiple myeloma can be diagnosed based on blood or urine tests finding abnormal antibodies, bone marrow biopsy finding cancerous plasma cells, and medical imaging finding bone lesions. Another common finding is high blood calcium levels. Multiple myeloma can be treated with e.g. steroids, chemotherapy, targeted therapy, and stem cell transplant.

Acute Myeloid Leukemia

[0058] AML is the most common form of acute leukemia in adults. It is characterized by infiltration of the bone marrow, blood, and other tissues by proliferative, clonal, abnormally differentiated, and occasionally poorly differentiated cells of the hematopoietic system. The median age of diagnosis is 68 years and incidence increases with age. The prognosis of patients diagnosed with AML is poor; varying from a 5 year overall survival of 40-50% in younger (<50 years) patients with de novo AML to ~ 5-10% in older patients, patients with secondary AML, or relapsed/refractory disease. The mainstay of AML therapy for relatively young <60 year and medically fit patients consists of intensive induction chemotherapy (traditionally anthracycline and cytarabine) followed by consolidation chemotherapy or allogeneic hematopoietic stem cell transplant. For patients that are not eligible for intensive regimens, therapy includes best-supportive care, low-dose cytarabine and hypomethylating agents decitabine and azacitidine. In case of relapsed or refractory AML, patients are offered intensive salvage therapy with the aim of achieving a complete remission and subsequent allogeneic hematopoietic stem cell transplant when deemed physically sufficiently fit. In other cases, patients receive low-intensity therapy or best supportive care.

[0059] Acute myeloid leukemia (AML) is a cancer of the myeloid line of blood cells, characterized by the rapid growth of abnormal cells that build up in the bone marrow and blood and interfere with normal blood cell production. Symptoms may include feeling tired, shortness of breath, easy bruising and bleeding, and increased risk of infection. Occasionally, spread may occur to the brain, skin, or gums. As an acute leukemia, AML can progress rapidly, and can be fatal within weeks or months if left untreated.

Risk factors for AML can include smoking, previous chemotherapy or radiation therapy, myelodysplastic syndrome, and exposure to the chemical benzene. The underlying mechanism involves replacement of normal bone marrow with leukemia cells, which results in a drop in red blood cells, platelets, and normal white blood cells. Diagnosis of AML can be based on bone marrow aspiration and specific blood tests. AML has several subtypes for which treatments and outcomes may vary. The first-line treatment of AML can be chemotherapy, with the aim of inducing remission. Patient may then go on to receive additional chemotherapy, radiation therapy, or a stem cell transplant. The specific genetic mutations present within the cancer cells define the AML subtype, may guide therapy, as well as determine how long that person is likely to survive. Before the diagnosis based on genetic aberrations (after 2016), AML subtypes were classified according to the French- American- British (FAB) MO - M7 classification, although the FAB classification can still be used. AML M4 (acute myelomonocytic leukemia) and AML M5 (acute monoblastic leukemia) are AML subtypes related to the monocyte lineage and are often expressing CD Id.

CD Id

[0060] CD Id is a member of the CD1 (cluster of differentiation 1) family of glycoproteins (including CD la, CD lb, CDlc, CD Id and CDle) and is expressed on the surface of various human cells, including certain antigen presenting cells (APC). They are non-classical MHC proteins, related to the class I MHC proteins, and are involved in the presentation of lipid antigens to a subgroup of T cells. In human CDld is encoded by CD1D, also known as R3G1. APC displaying CDld include B-cells, certain dendritic cells (e.g. in lymph nodes), and monocytes. CDld is also expressed by various other cell types, for example, in liver, pancreas, skin, kidney, uterus, conjunctiva, epididymis, thymus and tonsil (Canchis et al. (1992) Immunology 80:561).

[0061] Cells that are activated/stimulated via CDld include CDld restricted Natural Killer T-cells (NKT cells). NKT cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. NKT cells are a subset of T cells that express an alpha(a)/beta(P) T-cell receptor (TCR), as well a variety of molecular markers that are typically associated with NK cells. Type 1 or invariant NKT (iNKT) cells are the best-studied group of NKT cells and differ from conventional a0- T cells in that their T-cell receptors are far more limited in diversity ('invariant'). These invariant, but also other CD Id-restricted, NKT cells (type II NKT) recognize several antigens, such as (self or foreign) lipids, glycolipids, sulfatides, phospholipids, lipopeptides, hydrophobic peptides and/or amphipathic a-helical peptides, presented by CDld molecules present on APC (Enrico Girardi et al. (2016) J Biol Chem. 291(20): 10677). The interaction between (antigen-presenting) CDld and TCR triggers the release of cytokines including Thl- and/or Th2-like cytokines, such as interferon-y, tumor necrosis factor-a, and interleukins like IL-4, IL-5 and IL- 13.

[0062] Important roles of iNKT cells have been demonstrated in the regulation of autoimmune, allergic, antimicrobial, and antitumor immune responses (reviewed by van der Vliet et al. (2004) Clin Immunol 112(1): 8). Physiologically, the NKT-cells can augment or inhibit immune responses, including antitumor, autoimmune, and anti-pathogen responses, through a variety of mechanisms depending on context (Yue et al. (2010) J Immunol 184: 268), including induction of cell death in multiple myeloma cells. Conditions in which (invariant) NKT-cells may be involved include autoimmune or inflammatory diseases, including myasthenia gravis, psoriasis, ulcerative colitis, primary biliary cirrhosis, colitis, autoimmune hepatitis, atherosclerosis, and asthma. In addition to cytokine release, NKT cell effector functions which result in cell lysis, such as perforin release and granzyme release and cell death, may also be relevant in conditions in which NKT cells are implicated, such as in cancer.

[0063] Based on their T cell receptor (TCR) usage and antigen specificities, CD Id-restricted NKT cells have been divided into two main subsets: type I NKT cells that use a canonical invariant TCR a- chain and recognize a-galactosylceramide (a-GalCer), and type II NKT cells that use a more diverse aP-TCR repertoire and do not recognize a-GalCer. In addition, a-GalCer-reactive NKT cells that use non-canonical a0-TCRs and CD Id-restricted T cells that use y5- or 5/a0-TCRs have recently been identified, revealing further diversity among CD 1 d-restricted T cells (Macho -Fernandez and Brigl (2015) Front Immunol 6:362). [0064] The discovery of the first CD Id-presented antigen, a -galactosylceramide (a-GalCer), by Kawano and colleagues in 1997 enabled several important steps forward in our under-standing ofNKT cell biology, particularly of type I or invariant NKT (iNKT) cells (Kawano et al. (1997) Science 278: 1626). iNKT cells express an invariant Va24 Jal 8 a chain in humans, paired with V011 TCR 0 chain in humans). Type I NKT cells can be highly autoreactive even at steady state and display an activated/memory phenotype with high surface levels of the activation markers CD69, CD44, and CD 122 (IL-2R 0-chain) and low expression of CD62L, a marker expressed by naive T cells that home to lymph nodes (Bendelac et al (1992) J Exp Med 175:731; Matsuda et al. (2000) J Exp Med 192:741). Type I NKT cells critically contribute to natural anti-tumor responses, as demonstrated by the prompt growth of spontaneous tumors in type I NKT cell-deficient Jal 8-/- mice compared to WT mice (Smyth et al. (2000) J Exp Med 191 :661; Swann et al (2009) Blood 113:6382; Bellone et al (2010) PLoS One 5:e8646). Furthermore, the activation of type I NKT cells by a-GalCer provides potent effects against hematologic malignancies and solid tumors through their IFN-y-production and the subsequent activation of dendritic cells (DC) and NK cells (Smyth et al (2002) Blood 99: 1259; Berzofsky and Terabe (2008) J Immunol 180:3627). By contrast, sulfatide-activated type II NKT cells repress anti -tumor immunity (Terabe et al (2000) Nat Immunol 1 :515; Terabe et al (2005) J Exp Med 202: 1627; Renukaradhya et al (2008) Blood 111 :5637) by abrogating type I NKT activation in response to a-GalCer, in terms of cytokine secretion and expansion (Ambrosino et al (2007) J Immunol 179:5126). Moreover, their IL-13 production, in combination with TNF-a, led to upregulation of TGF-0 secretion by myeloid-derived suppressor cells (MDSC), and resulted in decreased cytotoxic T cell activity (Terabe etal (2003) J Exp Med 198:1741).

[0065] CD Id-restricted T cells that do not express the Val4-Jal 8 rearrangement and do not recognize a-GalCer were first described in MHC Il-deficient mice among the remaining CD4+ T cells (Cardell et al (1995) J Exp Med 182:993). From then called diverse NKT (dNKT), type II NKT, or variant NKT (vNKT) cells, this NKT cell population, found in both mice and humans, exhibits a more heterogeneous TCR repertoire. Several lines of evidence suggest that type II NKT cells can contribute to and modulate a range of immune responses, occasionally in opposing roles to type I NKT cells.

[0066] In addition to CD Id-restricted T cells with a0 TCRs, CD Id-restricted T cells expressing y5 TCRs have recently been described in both mice and humans. According to their V5-chain expression, human y5 T cells can be divided into two major populations: V52+ and “non-V52” subsets, the latter comprise, inter alia, V51+y5 T cells and the less prevalent V53+y5 T cells (McVay et al (1999) Crit Rev Immunol 19:431; Vantourout and Hayday (2013) Nat Rev Immunol 13:88). V51+y5 T cells are mainly tissue resident and are found in the skin and at mucosal surfaces, whereas V52+y5 T cells are predominant in human blood. Compared with T cells, the types of antigens recognized by y5 T cells and the role and function of antigen presentation in y5 TCR recognition are much less clear. Interestingly, some y5 T cells have recently been found to directly recognize CD Id-presented lipid antigens (Hayday and Vantourout (2013) Immunity 39:994). In 2013, Uldrich et al (2013) Nature Immunol 14: 1137) had identified a y5-T cell population that recognized CDld in combination with select glycolipid antigens, including aGalCer. These T cells are referred to as CD Id-restricted V51+ T cells. Without being bound to theory, it is believed that V51+ T cells can exert an inflammatory response that can be counterproductive for an effective anti-tumor response.

[0067] The majority of gammadelta peripheral blood lymphocytes (PBLs) in human adults express T- cell receptors (TCRs) comprising Vy9 and V52 regions. Vy9V52 T cells can react against a wide array of pathogens and tumour cells. This broad reactivity is understood to be conferred by phosphoantigens which are able to specifically activate this T-cell subset in a TCR dependent fashion. The broad antimicrobial and anti-tumour reactivity of Vy9V52 T-cells suggest a direct involvement in immune control of cancers and infections. Hence, agents that can activate Vy9V52T cells can be useful in the treatment of infections or cancer as these may promote Vy9V52T T cell reactivity towards the pathogen or infected cells or cancer.

Flow Cytometry

[0068] Flow cytometry is known and described in, for example, U.S. Pat. Nos. 2,656,508; 2,869,078; 3,271,671; 5,915,925; 6,248,590; 6,524,860; 6,589,792; 6,604,435; and 6,890,487. Most preferably detection is performed by incubating the labeled antibody with a plurality of cells after which unbound or non-specifically bound labeled antibody is washed away. Detection may be performed with a directly-labeled antibody or by first incubating with an unlabeled (primary) antibody which is detected by a secondary labeled anti-antibody. The labeling compound most preferably is a fluorescent compound such as a fluorochrome. Flow cytometry techniques (including fluorescent activated cell sorting or FACS) may be carried out in accordance with known techniques or variations thereof.

[0069] The choice of markers in diagnostic applications requires consideration of a variety of factors. The useful brightness of a dye depends on issues including (1) fundamental photophysical parameters (the product of the molar absorption coefficient at the wavelength of illumination (Uxc) and the fluorescence quantum yield of non-aggregated species; the separation of absorption and emission bands; photostability, etc.), (2) The fluorescence intensity and its decay in time, and (3) the photon flux at the sample (which depends on wavelength and absorption by any interfering species.

[0070] In addition to brightness, consideration also can be given to sensitivity and selectivity over background or other emitting species. Such considerations can be important in multicolor applications, where more than one dye marker may be employed yet spectral and/or lifetime discrimination is essential. Another practical factor concerns the industry-installed base of diagnostic instruments, which may have certain excitation sources and detectors. There are commercial advantages of dyes with superior chemical and conjugating attributes yet have spectral features that match the installed base of diagnostic instrumentation. In some cases, a sizable spacing between the absorption band (for excitation) and the emission band (for detection) is desired, so as to avoid interference from light scattering by the sample. In some cases, relatively sharp absorption and emission bands are desired, so that a large number of dye markers can be used in a given spectral region.

[0071] Cells may be fixed and permeabilized before they are brought into contact with detection antibodies. Alternatively, cells can be stained with one or more detection antibodies before they are permeabilized and further stained with additional detection antibodies. For example, in some embodiments, one or more detection antibodies recognizing cell surface biomarkers are used. It may be possible to stain cells with those detection antibodies before cells are permeabilized. The cells may then be permeabilized and further stained with other detection antibodies, such as those that recognize biomarkers expressed in the cytoplasm or the nucleus.

[0072] Generally, any agent for fixing and/or permeabilizing cells can be used, as long as it adequately preserves the cells of interest and it does not exhibit significant fluorescence at the emission wavelength of the fluorochrome or the autofluorescence. Suitable agents include, for example, 100% methanol, 4% paraformaldehyde followed with saporin detergent, 95% ethanol containing 2-5% polyethyleneglycol, PreservCyt (Cytyc Corp., Boxborough, Mass.), Cytorich (AutoCyt, Burlington, N.C.), and others known to those skilled in the art.

[0073] In some embodiments, the agents for fixation and/or permeabilization can be washed away from the cells before staining with detection antibodies. For example, after fixation, the cells can be sedimented to form a pellet and resuspended in a medium such as PBS. This process can be repeated several times if necessary.

[0074] In some embodiments, the cells are subject to a blocking step prior to staining to suppress nonspecific binding and to increase the sensitivity of the signal. Numerous methods for blocking nonspecific binding are known to those skilled in the art and are suitable. Exemplary blocking agents include a dilute (such as 2%) solution of a protein such as bovine serum albumin, casein, fetal bovine serum, and fish skin gelatin.

[0075] Methods of staining samples with detection antibodies are known in the art. Generally, the steps involve contacting the sample with one or more detection antibodies. In cases wherein the detection antibodies are not labeled prior to the contact, the sample can be further subject to a labeling step. The staining conditions (such as concentrations of various agents, temperature of the staining, etc.) are known in the art, and depend on the nature of the detection antibodies, the condition of the sample, and the biomarker to be detected. In some embodiments, the sample is pre-analyzed to determine the condition of the sample (e.g., how many cells are in the sample), and the staining condition can be adjusted accordingly. In some embodiments, the unbound detection antibodies are removed before the sample is further analyzed in a detecting instrument.

[0076] In embodiments when more than one detection antibody is used, the different detection antibodies may be added sequentially or simultaneously to the sample. In some embodiments, a mixture of probing agents (such as an antibody cocktail) is made. Optimal concentrations can first be determined for each detection antibody individually, and the detection antibodies at optimal concentration can then be mixed together. The cocktail of detection antibodies can be evaluated the same away as described above. The staining result can be compared to the staining result of each individual detection antibody to determine whether the detection antibodies in the cocktail function independently and do not interfere with one another. The sensitivity and specificity of the staining can also be evaluated.

[0077] The reagents making up the antibody cocktail can be provided as a pre-combined composition or can be combined before the staining (generally, but not necessarily just before staining). For example, in one embodiment, the cocktail can be provided as a single composition including each of the reagents. In other embodiments, some, but not all, of the detection antibodies are provided in a pre-mixed cocktail. In some embodiments, the cocktail can be provided as separate containers for each of reagent. Mixing of the reagents can be done right before the staining experiment, for example by a person or a machine such as a device described herein.

[0078] In some embodiments, one or more detection antibodies comprising an anti-CDld antibody is used for the staining. The antibodies are initially typically titrated to determine a concentration for optimal use. In some embodiments, the concentration of the CDld antibody is about 0.01 pg/ml to about 100 pg/ml. In some embodiments, the concentration of the CDld antibody is at least about 0.01 pg/ml. In some embodiments, the concentration of the CDld antibody is at most about 100 pg/ml. In some embodiments, the concentration of the CDld antibody is about 0.01 pg/ml to about 0.1 pg/ml, about 0.1 pg/ml to about 1 pg/ml, about 1 pg/ml to about 10 pg/ml about 10 pg/ml to about 100 pg/ml. The person skilled in the art may determine the optimal range for titration by first assessing the (apparent) KD of the antibody and titrate in the range of 0. IxKD to 1 OOxKD.

[0079] Detection may be carried out by any method that can measure (or determine the presence or absence of) a given parameter. In some embodiments, the detection instrument for detecting multiple parameters comprises a flow cytometer. In some embodiments, the detection instrument for detecting multiple parameters is part of a cell screening apparatus described herein.

Treatment of CD ld-expressing Tumor Cells

[0080] As described above, the method of the disclosure includes a treatment step wherein a prescreened subject whose sample exhibits a CD Id Fluorescence Index of at least 1.1 (e.g. at least 1.5) is treated with a bispecific antibody that comprises a binding moiety that binds CD Id and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var.

[0081] Bispecific antibodies that comprise a binding moiety that binds CD Id and a binding moiety that binds a Vy9V52-T cell receptor, such as antibody lD12-5C8var may potentially provide a therapy for CD ld-expressing tumors. As described, antibody lD12-5C8var consists of two VHH domain antibody fragments linked via a 5 -amino acid glycine-serine linker. One arm recognizes the V52 chain of the y5-T cell receptor (TCR) and thereby targets Vy9V52-T cells, the other arm is specific for the tumor antigen CDld. Antibody lD12-5C8var is disclosed in PCT Published Patent Application No. 2020060405 as SEQ ID NO:87 (SEQ ID NO:1 herein). WO 2020060405 also discloses the preparation of antibody lD12-5C8var and related antibodies, methods of formulating pharmaceutical compositions comprising antibody lD12-5C8var, methods of treating diseases, including CLL, MM, and AML, with antibody lD12-5C8var and related antibodies, and dosing and dose regimens using pharmaceutical compositions that comprise antibody lD12-5C8var. WO 2020060405 and related application WO 2020060406 are herein incorporated by reference in its entirety for all purposes.

Firstly, the anti-CDl d VHH recognizes CDld expressing tumor cells, while the other VHH targets the Vy9V52 TCR and thereby the molecule cross-links Vy9V52-T cells to tumor cells resulting in the activation of the former. This leads to degranulation of the Vy9V52-T cells, the secretion of cytolytic molecules and the subsequent death of the cancer cell. In addition, antibody lD12-5C8var has a dual mechanism of action in that it is also able to induce iNKT cell activation via binding to CDld and stabilization of the interaction between CDld and the invariant TCR of iNKT cells. Activated iNKT cells can exert direct cytotoxicity against CD Id-positive tumor cells and, in addition, produce various cytokines that promote the activity and cytotoxic potential of other immune cells (including Vy9V52- T cells) to induce subsequent CDld positive tumor cell lysis.

EXAMPLES

Example 1

Determination of biomarker expression levels using flow cytometry [0082] To assess if a patient is eligible for a treatment that targets CD 1 d, the expression level of CD 1 d on tumor cell subsets is assessed by means of calculating the CD Id fluorescence (Molecules of Equivalent Soluble Fluorochrome or MESF) ratio between a test sample treated with the fluorochrome-labelled CD Id antibody and a fluorochrome-labelled isotype control antibody-treated sample. The assay is carried out to determine the expression levels of CD Id on tumor cells in CLL patients, MM patients and AML patients.

Isolating mononuclear cells

[0083] First, a blood sample or bone marrow sample is obtained from the patient. Mononuclear cells, including peripheral blood mononuclear cells (PBMCs), or bone marrow mononuclear cells (BMMCs) are isolated from the patient blood or bone marrow sample, respectively, using Ficoll-Paque gradient separation following the steps below.

1) Invert the Ficoll-Paque medium bottle several times to ensure thorough mixing. Remove the snap- off polypropylene cap and withdraw the required volume of Ficoll-Paque medium by syringe or pipette. 2) Add Ficoll-Paque medium (3 mL) to the centrifuge tube. 3) Carefully layer the diluted blood sample (6 mL) onto the Ficoll-Paque medium solution. 4) Centrifuge at 400 g for 20 to 30 min at 18 °C to 20 °C (brake should be turned off). 5) Draw off the upper layer containing plasma and platelets using a sterile pipette, leaving the mononuclear cell layer undisturbed at the interface. The upper layer, which contains the plasma and platelets, may be saved for later use. 6) Transfer the layer of mononuclear cells to a sterile centrifuge tube using a sterile pipette, wash with an excess of phosphate buffered saline (PBS) solution and centrifuge the isolated mononuclear cells for 10 minutes at 120 x g to remove the platelets and Ficoll-Paque medium (repeat the washing procedure 2 times). Erythrocytes are subsequently lysed by ammonium chloride solution treatment for 10 minutes at 4 °C, and the mononuclear cell sample is washed with an excess of PBS. In some cases, the biological sample is a whole blood sample or a bone marrow sample, and erythrocytes are lysed by adding ammonium chloride solution at a ratio of 4:l(volume:volume) for 10 minutes at 4°C, and the cell sample is washed with an excess of PBS.

Antibody cocktail

[0084] Prior to the staining of cells, a white blood cell count (WBC count) is carried out on the Sysmex XS-lOOOi hematology analyzer or any alternative cell counting method known to a person skilled in the art and the appropriate cell concentration is adjusted.

[0085] Next, l*10^A6 mononuclear cells are equally divided over 2 tubes, and subsequently labeled with an antibody cocktail for the identification of the tumor cells (specific per disease) and additionally labeled with either an antibody detecting CD Id or an isotype control antibody (both labeled with the same fluoro chrome).

[0086] In one example, the antibodies included in each assay are CD45 (e.g., APC-Cy7, BD 557833), CD19 (e.g., PE-Cy7, BD 560728), CDld (e.g., BV421, BD 743604) or isotype (e.g., BV421, BD 562438) antibodies. Disease specific antibodies included in the B-CLL assay are CD23 (e.g., BV605, BD 743428), CD43 (e.g., BUV737, BD 749228), and CD5 (e.g., PerCP-Cy5.5, BD 341109) antibodies. Disease specific antibodies included in the MM assay are CD38 (e.g., BV605, BD 562665), CD138 (e.g., BV786, BD 743501), and CD56 (e.g., BUV737, BD 748609) antibodies. Disease specific antibodies included in the AML assay are CD117 (e.g., R718, BD 752296), CD13 (BV786 BD 744748), CD133 (BV605 BD 747643), CD34 (BV805 BD), CD7 (Pe-Cy7 BD 564019), CD22 (BV786 Pe-Cy7 BD 563941), and CD33 (PE-CF594 BD562492) antibodies. A viability dye (e.g. BV510) can also be present.

[0087] In another example, the antibodies included in the CLL assay are CDld or isotype, CD45, CD19, CD5, CD43, CD79b and CD23 antibodies. The antibodies included in the MM assay are CDld or isotype, CD45, CD19, CD38, CD138, and CD56 antibodies. The antibodies included in the AML assay are CDld or isotype, CD45, CD34, CD117, CD133, CD13, CD33, CD7, CD22 and CD19 antibodies. A viability dye (e.g. BV510) is also present. In some cases, the CD7, CD22, and CD19 antibodies may be linked to the same fluorochrome.

Flow cytometry and data analysis

[0088] Samples are analyzed with a flow cytometer (LSRFortessa X-20) equipped for measuring FITC, APC, PE, PerCP-Cy5.5, BV421/510/605/711/786, BUV395/737/805, PE-CF594, PE-Cy7, APC-Cy7 and R718. For acquisition, BD FACSDiva Flow Cytometry Software v8.0.2 was used. For data processing, De Novo FCSExpress 6 Flow Clinical Edition was used.

[0089] Stable performance of the flow cytometer is ensured by applying setup beads with stable fluorescence intensity through time of study. Each day a validation run is performed, setup beads are measured. BD Cytometer Setup and Tracking (CS&T) beads are used withBD FACSDiva acquisition software v8.0.2.

[0090] Compensations are performed with the BD Comp Beads for CD markers and ArcTM Amine Reactive Compensation Beads for live/dead staining. Compensation settings (percentages) are acquired before each day of acquisition.

[0091] To calculate Molecules of Equivalent Soluble Fluorochrome (MESF) values, fluorescence quantitation beads (e.g. SPHERO™ Ultra Rainbow Fluorescent Particles) are used. Fluorescence quantitation beads contain calibrated amounts of fluorescent molecules and hence have known MESF values. The fluorescence quantitation beads are measured prior to acquisition of the samples, and as a part of each run. The fluorescence quantitation bead MFI values, generated by the flow cytometer, are plotted against MESF units to generate a calibration curve. The calibration curve is used to calculate the MESF values of the samples by interpolation of the MFI generated by the flow cytometer.

[0092] The following reportables are analyzed and reported gated from viable and single cells:

• CDld MESF ratio on the appropriate tumor cell population of the disease (and optionally on monocytes/B cells): o CDld⁺ MESF; o Ratio calculated as MESF CD 1 d⁺ / MESF Isotype.

• Tumor cell subsets: o CLL: CD45+ / CD19+ / CD5+/- / CD79b+/- / CD23+/- / CD43+/- o MM: CD451ow/neg / CD19+/- / CD38+ / CD138+ / CD56+/- o AML: CD451ow/int / CD34+/- / CD117+/- / CD133+/- / CD13+/- / CD33+/-/ CD22+/-

/ CD7+/- / CD19+/-

“+” indicates that cells positive for the marker are selected through gating

“+/-“ indicates that tumor cells can be positive or negative for the marker (depending on the tumor cell phenotype) and that either positive or negative cells are selected through gating “low/neg” indicates that tumor cells can have low expression levels of the marker or are negative and are selected through gating

“low/int” indicates that tumor cells can have intermediate or low expression levels of the marker and are selected through gating

[0093] The validation experiments were carried out and a CD-Chex Plus whole blood control tube (QC) was included in each run to verify the antibody staining. In case an epitope for a specific antibody is not present in the CD-Chex Plus whole blood control tube, a cell line known to express that specific antigen can be included for QC. An BV421 CDld and BV421 isotype control antibody was used for CDld-MFI determination and calculation of MESF ratios. No acceptance criteria apply to the isotype controls

[0094] The samples are analyzed by flow cytometry and the acquired Molecules of Equivalent Soluble Fluorochrome (MESF) on the tumor cells of CDld are divided by the MESF of the isotype control, in order to calculate the MESF Index.

[0095] Analysis of the MESF Index on tumor cells was performed by gating the viable cells using the FSC/SSC, and subsequently the single- and viability dye negative cells in De Novo FCSExpress flow cytometry software. Next, the tumor cells were selected by gating the cells which do or do not express the appropriate tumor cell markers. FIG.l provides an illustration of the gating process for a sample from a CLL patient.

Example 2

Antibody-mediated tumor cell lysis by Vy9V82-T cells in patient samples containing CDld- expressing cells

[0096] The relation between CD Id expression and the ability of a bispecific CDld-Vy9V52-T cell engaging antibody to mediate tumor cells lysis by Vy9V52-T cells was tested as follows:

[0097] Vy9V52-T cells were isolated from PBMCs of healthy donors by Magnetic Activated Cell Sorting (MACS) isolation using an antibody targeting V52, and subsequently expanded by coculturing at a 1 :2 ratio with y-irradiated PBMCs from 2 healthy donors, in the presence of the cytokines interleukin (IL)-7 and IL-15. Vy9V52-T cells are cocultured for 7 - 14 days, during which medium is removed (while leaving the cell clusters intact) every 2 to 3 days and replenished with medium containing IL-7 and IL-15. Vy9V52-T cells were passaged for a maximum of 8 passages. Tumor cell lysis was assessed by culturing 5 *10^A4 peripheral blood mononuclear cells (PBMCs) from CLL patients, or bone marrow mononuclear cells (BMMCs) from MM-, or AML patients, at a 2:1 ratio with Vy9V52-T cells. After overnight coculture in the presence or absence of a single dose (10 - 50 nM) of antibody lD12-5C8var, the absolute number of viable (7AAD-negative) CLL, MM or AML cells was determined by flow cytometry by the adding counting beads to each tube before acquisition and normalizing each acquisition to a set number of counting beads. The relative tumor cell lysis within the patient’s cell sample following the coculture in presence of antibody lD12-5C8var, was calculated as a percentage increase over the coculture in absence of the TCE (set to 0%).

[0098] Analysis of a number of patient samples showed that Vy9V52-T cell mediated tumor cell lysis in the presence of bispecific antibody was consistently observed in patient samples displaying a CD Id MESF index of > 1.5 (see Table 1 and FIG. 2). Thus, patients with a CDld expression level (Fluorescence or MESF index) of >1.1 or > 1.5 measured on the patient tumor cells can be eligible for antibody therapy. Using the MESF index can allow robust assessment of the CDld expression level independent of flow cytometer settings, and in a cross-platform manner.

Table 1

[0099] While specific embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. It should be understood that various alternatives to the embodiments of the present disclosure may be employed. [0100] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application herein is not, and should not be, taken as acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1. A method of treating a subject with a hematological cancer, the method comprising:

I. a prescreening step comprising: i. providing a biological cell sample comprising mononuclear cells, wherein said sample is obtained from a subject with a hematological cancer; ii. labeling a first portion of cells via (a) an antibody that binds CD Id and (b) one or more antibodies that bind to one or more other antigens that are known to be expressed, or known to be absent, on tumor cells in subjects with said hematological cancer; iii. labeling a second portion of cells via (a) an isotype control antibody and (b) the same antibodies as in ii. (b) above; iv. analyzing the labelled cells using flow cytometry; v. selecting the flow cytometry data set corresponding to tumor cells on the basis of the signal generated by the antibodies specified in (b), and vi. calculating a CD Id Fluorescence Index from the acquired fluorescence for tumor cells labeled via the antibody that binds CD Id and the acquired fluorescence for tumor cells labeled via the isotype control antibody; and

II. a treatment step wherein a prescreened subject whose sample exhibits a CD Id Fluorescence Index of at least 1.1 is treated with a bispecific antibody that comprises a binding moiety that binds CD Id and a binding moiety that binds a Vy9V52-T cell receptor, for example antibody lD12-5C8var.

2. The method of claim 1 , wherein the hematological cancer is selected from chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and multiple myeloma (MM).

3. The method of claim 1 or 2, wherein the sample consists essentially of peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs) obtained from a subject diagnosed with CLL.

4. The method of claim 1 or 2, wherein the sample consists essentially of PBMCs or BMMCs obtained from a subject diagnosed with AML.

5. The method of claim 1 or 2, wherein the sample consists essentially of PBMCs or BMMCs obtained from a subject diagnosed with MM.

6. The method of claim 3, wherein PBMCs or BMMCs are divided into two samples comprising approximately equal numbers of PBMCs or BMMCs, which are labeled as follows: I. PBMCs or BMMCs from the first sample are labelled with antibodies comprising (a) an antibody that binds CD Id conjugated to a fluorochrome and (b) one or more antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with CLL; and

II. PBMCs or BMMCs from the second sample are labelled with antibodies comprising (a) an isotype control antibody conjugated to the same fluorochrome used in I. above and (b) the same antibodies used in I. above that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with CLL.

7. The method of claim 4, wherein PBMCs or BMMCs are divided into two samples comprising approximately equal numbers of PBMCs or BMMCs, which are labeled as follows:

I. PBMCs or BMMCs from the first sample are labelled with antibodies comprising (a) an antibody that binds CD Id conjugated to a fluorochrome and (b) one or more antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with AML; and

II. PBMCs or BMMCs from the second sample are labelled with antibodies comprising (a) an isotype control antibody conjugated to the same fluorochrome used in I. above and (b) the same antibodies used in I. above that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with AML.

8. The method of claim 5, wherein BMMCs or BMMCs are divided into two samples comprising approximately equal numbers of BMMCs or BMMCs, which are labeled as follows:

I. BMMCs or BMMCs from the first sample are labelled with antibodies comprising (a) an antibody that binds CD Id conjugated to a fluorochrome and (b) one or more antibodies that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with MM; and

II. BMMCs or BMMCs from the second sample are labelled with antibodies comprising (a) an isotype control antibody conjugated to the same fluorochrome used in I. above and (b) the same antibodies used in I. above that bind to one or more other antigens that are known to be expressed or known to be absent on tumor cells in subjects with MM.

9. The method of claim 6, wherein the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with CLL specifically bind to CD5, CD19, CD23, CD43, CD45 and/or CD79b.

10. The method of claim 7, wherein the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with AML specifically bind to CD7, CD13, CD19, CD22, CD33, CD34, CD45, CD117 and/or CD133.

11. The method of claim 8, wherein the antibodies that bind to antigens that are known to be expressed or known to be absent on tumor cells in subjects with MM specifically bind to CD19, CD38, CD45, CD56 and/or CD138.

12. The method of any one of claims 6-11, wherein the one or more antibodies (excluding antibodies that bind CD Id and excluding isotype control antibodies) are conjugated to a fluorochrome that is different from the fluorochrome conjugated to antibodies that bind to CD1 d and isotype control antibodies.

13. The method of any one of claims 1-12, wherein the antibodies used in step ii. and iii. are directly conjugated to a fluorochrome.

14. The method of any one of claims 1-12, wherein the antibodies that bind the cells are detected via a secondary antibody which carries the fluorochrome.

15. The method of any one of claims 1 -14, wherein the fluorescence for mononuclear cells labeled with an antibody that specifically binds to CD Id and the fluorescence for mononuclear cells labeled with an isotype control antibody are calculated from the fluorescent light measurements collected by the flow cytometer.

16. The method of any one of claims 1-15, wherein the CD Id Fluorescence Index is a CDld MESF Index calculated from the acquired MESF for mononuclear cells labeled with an antibody that binds CD Id and the acquired MESF for mononuclear cells labeled with an isotype control antibody.

17. The method of any one of claims 1-16, wherein the MESF for mononuclear cells labeled with an antibody that specifically binds to CD Id (CD Id MESF) and the MESF for mononuclear cells labeled with an isotype control antibody (Isotype MESF) are used to calculate the CD Id MESF index according to the formula: CD Id MESF index = CDldMESF / Isotype MESF

18. The method of any one of claims 1-17, wherein a subject with CLL whose sample exhibits a CDld Fluorescence Index of at least 1.5 following screening is treated with antibody lD12-5C8var.

19. The method of any one of claims 1-17, wherein a subject with AML whose sample exhibits a CDld Fluorescence Index of at least 1.5 following screening is treated with antibody lD12-5C8var.

20. The method of any one of claims 1-17, wherein a subject with MM whose sample exhibits a CDld Fluorescence Index of at least 1.5 following screening is treated with antibody lD12-5C8var.

21. The method of any preceding claim, wherein the subject has relapsed or refractory CD Id- positive CLL, AML or MM.