ANTIBODIES TARGETING THE B-CELL RECEPTOR OF CHRONIC LYMPHOCYTIC LEUKEMIA AND USES THEREOF
20230272073 · 2023-08-31
Assignee
Inventors
Cpc classification
C07K2317/24
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention provides antibodies for the treatment of chronic lymphocytic leukemia (CLL). These antibodies target the B-cell receptor (BCR) of CLL cells characterised by R110-mutated immunoglobulin lambda variable 3-21 (IGLV3-21.sup.R110).
The invention also provides nucleic acid sequences encoding the forgoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.
Claims
1. Antibodies for use in the treatment of CLL in IGLV3-21.sup.R110 positive patients, wherein said antibodies have a heavy chain amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence of SEQ ID NO: 2; or a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO: 12; or comprise a variable heavy chain having a sequence selected from the list consisting of SEQ ID NO: 15 and SEQ ID NO: 20 in any combination with a variable light chain having a sequence selected from the list of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19.
2. Antibodies for use according to claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 1 and a light chain corresponding to SEQ ID NO: 2.
3. Antibodies for use according to claim 1, characterized by a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO: 12.
4. Antibodies for use according to claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 14.
5. Antibodies for use according to claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 21 and a light chain corresponding to SEQ ID NO: 14.
6. Antibodies for use according to claim 1, which are chimeric.
7. Antibodies for use according to claim 4, which are humanized.
8. Antibodies for use according to claim 1, wherein the antibodies are applied at a dose of from 0.25 to 25 mg/kg.sub.bodyweight.
9. Antibodies for use according to claim 8, wherein the antibodies are applied at a dose of from 1 to 20 mg/kg.sub.bodyweight.
10. Antibodies for use according to claim 8, wherein the antibodies are applied at a dose of from 7 to 15 mg/kg.sub.bodyweight.
11. Antibodies for use according to claim 8, wherein the antibodies are applied at a dose of from 8 to 12 mg/kg.sub.bodyweight.
12. A pharmaceutical composition for use in the treatment of CLL in IGLV3-21.sup.R110 positive patients comprising antibodies for use according to claim 1 and a pharmaceutically acceptable carrier or excipient.
13. A kit for use in the treatment of CLL in IGLV3-21.sup.R110 positive patients comprising a pharmaceutical composition for use according to claim 12.
14. Antibodies for use according to claim 3, which are chimeric.
15. Antibodies for use according to claim 5, which are humanized.
16. Antibodies for use according to claim 7, wherein the antibodies are applied at a dose of from 0.25 to 25 mg/kg.sub.bodyweight.
17. Antibodies for use according to claim 7, the antibodies are applied at a dose of from 8 to 12 mg/kg.sub.bodyweight.
18. Antibodies for use according to claim 15, wherein the antibodies are applied at a dose of from 0.25 to 25 mg/kg.sub.bodyweight.
19. Antibodies for use according to claim 15, wherein the antibodies are applied at a dose of from 1 to 20 mg/kg.sub.bodyweight.
20. Antibodies for use according to claim 15, wherein the antibodies are applied at a dose of from 8 to 12 mg/kg.sub.bodyweight.
Description
DESCRIPTION OF THE FIGURES
[0177]
[0178] Pursuant to Example 4 the plots in
[0179] As can be readily appreciated from
[0180]
[0181]
[0182]
[0183] From the comparison of the top left picture to all of the remainder pictures of the left column can be seen that the mAb01-01 positively and selectively identifies diseased B-cells in the spleen of a diseased patient and that there is no cross-reactivity to any of the healthy donor's samples neither in the same tissue (spleen), nor in any of the other tissue types (skin, kidney, heart and brain). This shows that the antibodies of the present invention are selective, safe and non-cross-reactive. The further pictures of the right column show that the staining achieved in the top left picture and also the lack of staining in lower pictures of the left column is not related to any background or other artificial effect.
[0184]
[0185]
[0186]
[0187]
[0188] The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
[0189] All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following examples can be carried out as de-scribed in standard laboratory manuals, such as Sambrook et al., 1989 supra.
[0190] A preferred embodiment of the invention is:
EXAMPLES
Example 1
[0191] Generation of Murine Antibodies
[0192] Immunization and Generation of Hybridoma Cell Line
[0193] A murine antibody to the IGLV3-21.sup.R110-harboring BCR was developed by a combination of immunization of mice with a soluble form of the BCR and selection of suitable antibodies using a cell system in which the complete and functional BCR was presented membrane bound.
[0194] At first, a soluble form of the BCR in the form of an IgG.sub.1 had to be obtained for immunization of mice. Therefore, a DNA segment encoding IGHV3-21 as an exemplary variable heavy chain (VH) and a complete light chain (LC) DNA covering IGLV3-21.sup.R110 were synthesized by a contract manufacturer using a standard procedure. These were then fused with a murine IgG.sub.1 constant segment by polymerase chain reaction (PCR) and cloned into a cytomegalovirus (CMV) vector. A human cellular expression system based on HEK293T cells was used for the expression of such IgG.sub.1 (SEQ ID NO: 49 for VH, and SEQ ID NO: 50 for LC) as previously described, e.g. in Rekombinante Antikörper, Lehrbuch und Kompendium für Studium und Praxis, 2. Auflage, Springer Verlag 2019. A polyethyleneimine (PEI) based protocol was used for transfection. After several passages, the supernatant was pooled and the medium contained in the combined cell supernatant was purified using Protein G columns. The purity and quality of the soluble IgG.sub.1 was determined by Western blotting.
[0195] Thereafter, mice were immunized with the recombinantly produced soluble form of the BCR (cf. SEQ ID NOS: 49 and 50).
[0196] Immune cells with the desired specificity could then be obtained from these mice and transformed into hybridoma cells by cell fusion. Second, FACS screening methods were performed with triple knockout cells (TKO; knockout for the genes Lambda5, RAG2 and SLP65) expressing various variants for the BCR to select for antibodies specifically targeting BCRs harboring IGLV3-21.sup.R110.
[0197] Monoclonal antibody was produced using the standard procedure in mice and the subsequent generation of hybridoma cells.
[0198] This approach allowed the isolation of the unique monoclonal antibody “mAb01-01” (SEQ ID NO: 1 for heavy chain, and SEQ ID NO: 2 for light chain).
[0199] Selection of Monoclonal Antibody
[0200] The screening for positive clones was not performed by enzyme linked immunosorbent assay (ELISA) as usual. Since the target structure is a membrane-bound receptor, it is of central importance to validate the binding of the potential antibodies in a cellular system, i.e. while largely preserving the cell physiological states native to this cell type. First, groups of pooled supernatants were examined for binding events using fluorescence activated cell sorting (FACS) analysis. For this purpose different BCR variants were expressed on the surface of a triple knockout (TKO) cell line, which cannot express BCR itself.
[0201] The starting point for the production of TKO cells is formed by transgenic mice which have a respective knockout for the genes Lambda5, RAG1 or RAG2 and SLP65 (Dühren von Minden et al., 2012, Nature 489, p. 309-313). The combination of the knockouts of RAG2 or RAG1 and Lambda5 leads to a blockade in the transition from the pro-B cell stage to the pre-B cell stage, which is classically characterized by the beginning rearrangement of the VDJ segments of the heavy chain (HC). Therefore they are pro-/pre-B cells. The activity of the BCR can be measured by reconstitution with the inducible SLP65. The production of such mice is known to the expert and belongs to the state of the art. To obtain the cells, the bone marrow of the femur was extracted from the mice after they had been sacrificed. The cells obtained in this way were then cultured under conditions that promote the survival of pro-/pre-B cells (37° C., 7.5% CO2, Iscoves medium, 10% FCS, P/S, murine IL7). After several passages, FACS sorting was carried out for control purposes, the pro-/pre-B cells were sorted and then returned to culture. The markers used for this purpose are known to the specialist.
[0202] For reconstitution with a ‘BCR of interest’, the corresponding sequence coding for the VH was fused with a human IgM constant segment by polymerase chain reaction (PCR), and heavy (HC) and light (LC) chains were cloned into respective expression vectors each having a CMV promoter. These were introduced into the packaging cell line (Phoenix cell line) by lipofection. After 36 hours of incubation, the virus supernatant was removed and used for Spinfektion of the TKO cells. BCR expression was determined using anti-IgM and anti-LC antibodies on FACS. For this purpose, some cells were taken and stained with 5p1 antibody each in a total volume of 100 μl in PBS. Both the work to extract the supernatants and the Spinfektion of the TKO are widely known procedures and known to experts. Knockout of RAG2 or RAG1 and Lambda5 ensured that only the “BCR of Interest” was expressed on the surface.
[0203] In this way, two different BCR-expressing TKO cell lines were generated, one of which expressed the membrane-bound IGHV3-21/IGLV3-21.sup.R110 BCR. For the generation of the second BCR-expressing TKO cell line, the codon for the arginine at position 110 of the DNA encoding IGLV3-21.sup.R110 was reverted to the germline sequence by well-known site-directed mutagenesis technique (see, e.g. Sambrook et al., 1989 supra). The resulting TKO cells expressed a BCR containing IGLV3-21 with glycine at amino acid position 110)(IGLV3-21.sup.G110). To generate a third control TKO cell line without BCR expression on its surface, spinfection with an empty expression vector was performed. By using an inducible SLP65 to reconstitute the cells, the function of the expressed BCRs could be characterized and the autonomously active state of the IGHV3-21/IGLV3-21.sup.R110 BCR on the surface could thus be verified before selection. The method of choice here is the measurement of Ca-flux after induction of SLP65 using FACS analysis and the use of a Ca.sup.2+ dependent dye such as Indo-1. These methods are known to the expert (see M. Dühren-von Minden et. al; Nature 2012).With these cells as “targets”, FACS has now been used to identify an antibody that specifically binds to IGLV3-21.sup.R110-harboring BCRs. The first step was to identify the supernatants whose antibodies showed a binding. In this 1st selection round, supernatants of several clones were combined and examined with regard to their binding profile. A positive binding profile is given if a specific binding to the IGHV3-21/IGLV3-21.sup.R110-BCR is shown. Groups showing such a profile were isolated, and the binding profile of the individual clones was characterized again during a second selection round. Binding of the monoclonal antibodies was verified using a FACS binding assay using a fluorescently labeled anti-mouse IgG antibody.
[0204] This selection approach led to the identification of the antibody mAb01-01, which binds the IGHV3-21/IGLV3-21.sup.R110 BCR positive TKO mouse cells, but not IGHV3-21/IGLV3-21.sup.G110 BCR positive TKO mouse cells.
[0205] Production of Murine Antibody
[0206] After identification of a preferred antibody by selection, mRNA was isolated from the individual hybridoma clone, cDNA was generated and amplified by Anchor PCR (Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination; Osterroth F, Alkan O, Mackensen A, Lindemann A, Fisch P, Skerra A, Veelken H. J Immunol Methods 1999 Oct. 29; 229(1-2):141-53). The sequence of the cDNA encoding the monoclonal antibody mAb01-01 was confirmed by Sanger sequencing (SEQ ID NO: 25 for HC nucleotide, SEQ ID NO: 26 for LC nucleotide) and placed into a vector suitable for expression in CHO cells.
[0207] Expression of the mAb01-01 as IgG1 subtype was verified using secondary anti-murine IgG1-APC and IgG2-APC antibodies. For this purpose, IGHV3-21/IGLV3-21.sup.R110-expressing TKO cells were stained in one batch with the secondary antibody alone and in another batch with mAb01-01 and the secondary antibody. Subsequent FACS analysis confirmed that the antibody had been expressed as IgG1.
[0208] The specific monoclonal antibody mAb01-01 was sequenced. The following amino acid sequences were determined as depicted in Table 1: SEQ ID NO: 1 for the HC, SEQ ID NO: 2 for the LC, SEQ ID NO: 3 for the VH, SEQ ID NO: 4 for the VL. The sequences corresponding to complementarity determining regions (CDR) of the heavy chain, H-CDR1, H-CDR2 and H-CDR3 are included in SEQ ID NOS: 5, 6 and 7, while the sequences corresponding to the light chain CDRs, L-CDR1, L-CDR2 and L-CDR3 are included in SEQ ID NOS: 8, 9 and 10.
Example 2
[0209] Generation of Chimeric Antibodies
[0210] Using the murine monoclonal antibody mAb01-01 VH and VL nucleotide sequences (SEQ ID NO: 27 for VH Nucleotide, and SEQ ID NO: 28 for VL Nucleotide) a chimeric antibody was synthesized. For this purpose, the VH sequence was fused with a human IgG1 isotype constant domain sequence (SEQ ID NO: 31 for IgG1 constant Nucleotide) and the VL sequence was fused with a human IgK isotype constant domain (SEQ ID NO: 32 for IgK constant Nucleotide) by PCR and expressed using a CHO based transient expression system. The resulting antibody containing cell culture supernatant was clarified by centrifugation and filtration. The chimeric antibody was purified from cell culture supernatant via affinity chromatography. The purity of the antibody was determined to be >95%, as judged by reducing and denaturing Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The antibody was analyzed for protein content and concentration via Seize Exclusion Chromatography (SEC) in PBS-buffer. All steps were performed with state-of the-art equipment and techniques.
[0211] This approach resulted in the chimeric antibody “HCO-LC0”, the sequences of which are summarized in Table 1.
TABLE-US-00001 TABLE 1 Sequences of murine and chimeric antibodies SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: VH VL HC Antibody HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Protein Protein Protein mAb01-01 5 6 7 8 9 10 3 4 1 HC0-LC0 5 6 7 8 9 10 3 4 11 SEQ SEQ SEQ SEQ SEQ SEQ ID NO: ID NO: ID NO: ID NO: ID NO: ID NO: LC VH VL HC LC HC + LC Antibody Protein Nucleotide Nucleotide Nucleotide Nucleotide Protein mAb01-01 2 27 28 25 26 39 HC0-LC0 12 27 28 29 30 40
Example 3
[0212] Generation of Humanized Antibodies
[0213] Humanization of mAb01-01 was carried out by in silico grafting the murine CDR's into mature human antibody frameworks using standard CDR-grafting technologies by Fusion Antibodies Plc, Belfast, N. Ireland. Key residues important for the VH/VL interface and canonical loop structure have been maintained as much as possible in the humanized variants using the CDRx platform (Fusion Antibodies Plc, Belfast, N. Ireland). Subsequently, the amino acid sequences of the humanized variants generated by fusion antibodies were converted to nucleotide sequences using Geneiouse software (Geneious Prime 2, Auckland, New Zealand). By fusing VH sequences with a human IgG1 isotype constant domain sequence (SEQ ID NO: 31 for IgG1 constant Nucleotide) and VL sequences with a human IgK isotype constant domain (SEQ ID NO: 32 for IgK constant Nucleotide) by PCR, 16 pairs of the humanized heavy and light chains were generated, and the antibody gene sequences expressed transiently in Chinese Hamster ovary cells (CHO). Following batch culture, expressed humanized antibodies were purified from the cell culture supernatant and analyzed as described in Example 2 for HC0-LC0. Eight humanized antibodies as depicted in Table 2 were successfully yielded.
TABLE-US-00002 TABLE 2 Sequences of humanized antibodies SEQ ID SEQ ID SEQ ID SEQ SEQ SEQ SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID Anti- NO: NO: NO: ID NO: ID NO: ID NO: NO: VH NO: VL NO: HC NO: LC NO: VH NO: VL NO: HC + LC body HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 Protein Protein Protein Protein Nucleotide Nucleotide Protein HC6-LC6 5 6 7 8 9 10 15 16 13 14 33 34 41 HC6-LC7 5 6 7 8 9 10 15 17 13 22 33 35 42 HC6-LC8 5 6 7 8 9 10 15 18 13 23 33 36 43 HC6-LC9 5 6 7 8 9 10 15 19 13 24 33 37 44 HC7-LC6 5 6 7 8 9 10 20 16 21 14 38 34 45 HC7-LC7 5 6 7 8 9 10 20 17 21 22 38 35 46 HC7-LC8 5 6 7 8 9 10 20 18 21 23 38 36 47 HC7-LC9 5 6 7 8 9 10 20 19 21 24 38 37 48
Example 4
[0214] Binding of mAb01-01 to IGLV3-21.sup.R110-BCR and IGLV3-21.sup.G110-BCR expressing TKO Cells
[0215] The specificity of the antibody mAb01-01 observed in the selection (Example 1) was verified in a FACS assay using the antibody coupled to a fluorescent marker.
[0216] For this purpose, from the three different TKO cell lines according to Example 1 two cell mixtures, cell mix A and cell mix B, were prepared and subsequently stained with mAb01-01-APC (mAb01-01 was coupled with fluorescent marker APC by Immunotools GmbH). For control, each batch was additionally stained with anti-IgM antibody (Anti-human IgM-PE, Clone: MHM-88, BioLegend Cat-No.: 314508).
[0217] Cell mix A was prepared at a 1:1 ratio of IGHV3-21/IGLV3-21.sup.R110 B-cell receptor expressing TKO cells and B-cell receptor negative (empty vector; control) TKO cells in PBS buffer (Gibco, pH 7.2, Cat. no. 20012-019).
[0218] Cell mix B was prepared at a 1:1 ratio of IGHV3-21/IGLV3-21.sup.G110 B-cell receptor expressing TKO cells and B-cell receptor negative (empty vector; control) TKO cells in PBS buffer.
[0219] Cells of cell mix A or B were suspended at approximately 106 cells per FACS tube in dilutions of the antibodies (5 μg/ml mAb01-01-APC, 2 μg/ml Anti-human IgM-PE, in a total volume of 100 μl) in PBS buffer, and incubated for 15 min at 4° C. in the dark. In the following, cells were washed once with 1 ml cold PBS buffer, resuspended in 200 μL cold PBS buffer.
[0220] FACS analysis was performed by using a MACSQuant Analyzer 10 (Miltenyi Biotec B.V. & Co. KG; the instrument was calibrated as recommended by the manufacturer, flow rate: Low, Mix sample: Mix gentle, Mode, Standard, Uptake volume: 50 μL, Sample volume: 200 μL). TKO-cells of cell mix A or cell mix B were gated in a side scatter (SSC) vs. forward scatter (FSC) and the gated TKO-cells were analysed in anti-IgM-PE vs. mAb01-01-APC dot-plot to enumerate the different TKO-cell populations using quadrant stats.
[0221] As shown in
Example 5
[0222] Affinity of Antibodies for the IGLV3-21.sup.R110 B-Cell Receptor
[0223] To define the binding affinities of the antibodies to the IGLV3-21.sup.R110-harboring B-cell receptor, a soluble recombinant version of the BCR (170.5 kDa; sequence according to SEQ ID NO: 51 for HC and 52 for LC) was produced in 293-HEK cell line as monomeric human IgM by transient expression using a protocol described in Example 1, and binding to immobilized anti-IGLV3-21.sup.R110 antibodies was monitored by Bio-Layer Interferometry (BLI) on a Fortebio Octet instrument (Satorius).
[0224] Kinetic assays were performed by first immobilizing the anti IGLV3-21.sup.R110 antibodies onto biosensors through an indirect capturing reagent, anti-human IgG Fc antibody. Anti IGLV3-21.sup.R110 antibodies were loaded at a concentration of 0.01875 μg/ml to generate an anti IGLV3-21.sup.R110 antibody capture level of between 0.30 and 0.34 nm. A 9 nM BCR-fragment solution in running buffer (PBS, 0.02% Tween20, 0.1% BSA, 0.05% sodium acide) was prepared and serial diluted 1:3 to obtain 7 concentrations from 9 to 0.012 nM (9 nM, 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, and 0.012 nM). The anti IGLV3-21.sup.R110 antibody capture biosensors were then submerged in wells containing the different concentrations of the soluble BCR-fragment for 900 seconds (association stage) followed by a dissociation step of 1200 seconds in running buffer. Steps were performed at a constant shake speed of 1000 rpm. All reagents were used as described by the manufacturer. Sensorgrams were generated after double reference correction (buffer and blank sensors) to compensate for both the natural dissociation of the capture anti IGLV3-21.sup.R110 antibody and also non-specific binding of the soluble BCR-fragment to the sensor surface. Dissociation rate constants (K.sub.D) were calculated based on the ratio of association (k.sub.a) and dissociation rate (k.sub.d) constants, obtained by fitting sensorgrams with a first order 1:1 binding model using the Fortebio Data Analysis software (Satorius).
[0225] As shown in Table 3, chimeric antibody HC0-LC0 binds with a K.sub.D value around 120 nM the soluble IGLV3-21.sup.R110 B-cell receptor. The humanized antibodies HC6-LC6 and HC7-LC6 exhibit binding characteristics similar to that of chimeric antibody HC0-LC0, exhibiting dissociation constants within 2-fold of the chimeric antibody HC01 LC01. For the K.sub.D values of all humanized antibodies, see Table 3.
TABLE-US-00003 TABLE 3 Monovalent K.sub.D values of chimeric antibody HC0-LC0 and humanized variants as measured by Fortebio with soluble IGLV3-21.sup.R110 B-cell receptor and anti IGLV3-21.sup.R110 antibody Capture levels K.sub.D Capture Antibody (M) level (nm) HC0-LC0 1.21E−10 0.322 HC6-LC6 2.11E−10 0.305 HC6-LC7 3.47E−10 0.337 HC6-LC8 2.77E−10 0.313 HC6-LC9 2.91E−10 0.311 HC7-LC6 1.75E−10 0.322 HC7-LC7 2.69E−10 0.323 HC7-LC8 2.49E−10 0.330 HC7-LC9 3.13E−10 0.280
Example 6
[0226] Binding of Murine Antibody mAb01-01 to Cell Surface of IGLV3-21.sup.R110-BCR Positive Human B-CLL Cells
[0227] To determine the binding characteristics of the mAb01-01 on IGLV3-21.sup.R110-B-cell receptor positive human CLL cells vs. non-IGLV3-21.sup.R110 human B-cells, binding was tested by flow cytometry.
[0228] For this purpose, cryopreserved peripheral blood mononuclear cells (PBMCs) of two CLL patients were used. The CLL in one of these patients was characterized by the presence of IGLV3-21.sup.R110. More specifically, the CLL cells expressed a BCR with a combination of IGLV3-21.sup.R110 with IGHV4-39 heavy chain (IGHV4-39/IGLV3-21.sup.R110-BCR). The other patient had been diagnosed suffering with a non-IGLV3-21.sup.R110 CLL. The PBMCs could be separated from heparinized venous blood by Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB) density gradient centrifugation using a technique that is known in the art, e.g. according to Bøyum A. Isolation of mononuclear cells and granulocytes from human blood. Scan. J. Clin. Lab. Invest. 1968, 21 (Suppl. 97): 77-89.
[0229] Samples where thawed and resuspended in 5 ml cell culture medium (RPMI, Gibco; 10% FCS, PAN-Biotec). Cells were centrifuged by 300 g (Eppendorf centrifuge 5425R), followed by additional resuspension in 1 ml RPM I. Cell count was acquired by using a Neubauer Chamber. For staining 1×10E6 cells were used and transferred in a FACS-tube. Cells were stained using 2p1 anti-CD19 VioBright515 (Miltiny Biotech, Klon:REA675), 2p1 Anti-CD5-PE-Vio770 (Miltenyi Biotec, Klon:REA782), and 5 μl mAb01-01-APC (mAb01-01 was coupled with fluorescent markers APC by Immunotools GmbH) in a total volume of 100 μl PBS buffer, and incubated for 15 min at 4° C. in the dark. In the following, cells were washed once with 1 ml cold PBS buffer, resuspended in 200 μL cold PBS buffer and analysed by flow cytometry using a BD LSRFortessa™ Cell Analyzer (BDbioscience). The instrument was calibrated as recommended by the manufacturer. The Analysis of the raw data were performed by using the FlowJo-Software X (BDbioscience). The analysis gates where set as demonstrated in
[0230] As shown in
Example 7
[0231] Binding Characteristics of Chimeric and Humanized Antibodies to Cell Surface of IGLV3-21.sup.R110-BCR Positive Murine TKO Cells
[0232] To compare the specific IGLV3-21.sup.R110-BCR binding of the chimeric antibody and two humanized versions, IGLV3-21.sup.R110-BCR murine TKO cells (see Example 1) were incubated with different concentrations of the antibodies HC0-LC0, HC6-LC6 and HC7-LC6 and analysed by flow cytometry and a sandwich assay setup. Controls were performed with a TKO—empty vector cell line (without surface BCR).
[0233] From each cell line, 7.5×10E6 cells are transferred into a separate 15 ml bluecap, centrifuged for 10 minutes (300 g, at 4° C.), and resuspended in 1.5 ml PBS (Gibco).
[0234] Staining where performed in a 96-well plate (VWR, U-bottom, non-treated). For each reaction 2×10E5 cells were used. The experimental setup is shown in Table 4.
TABLE-US-00004 TABLE 4 Experimental setup of the 96-well plate well No./Conc. (μg/ml) 1 2 3 4 5 6 7 8 9 10 1st Ab A: TKO-R110 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.00 HC0/LC0 B: TKD-emty vector 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.00 HC0/LC0 C: TKO-R110 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.00 0.00 HC7/LC6 D: TKO empty vector 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.00 HC7/LC6 C: TKO R110 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.00 HC6/LC6 D: TKO-empty vector 10.00 5.00 2.50 1.25 0.63 0.31 0.16 0.08 0.04 0.00 HC6/LC6
[0235] To characterize the binding characteristics of the different humanized variants 10 different concentrations of each antibody in PBS were used for staining (10, 5, 2.5, 0.625, 0.31, 0.16, 0.08, 0.04, 0 μg/ml) in a total volume of 200 μl per well. Incubation was performed for 30 min at 4° C., dark. The 96-well was then centrifuged (VWR, MEGA STAR 1.6R) for 10 min at 300 g, 4° C. Supernatants were discarded and the cells were resuspended in 100 μl ice cold PBS. For detection a secondary antibody directed to human IgG1 labeled with APC was used in a final concentration of μg/ml.
[0236] Incubation was performed in 200 μl/well total volume for 15 min at 4° C. in the dark, followed by an additional washing step with ice cold PBS. Cells were resuspended in 150 μl ice cold PBS for acquisition. Cells were analysed on a MACS-Quant10 (Miltenyi Biotec), calibrated followed by the instruction of the manufacturer.
[0237] MFI (median fluorescence intensity) of all IGLV3-21.sup.R110-BCR TKO measurements was neutralized by subtracting the control cell values and plotted against the concentration of the antibodies. Functions were generated demonstrating a concentration-dependent increase in binding of the IGLV3-21.sup.R110-BCR for all three antibodies. As shown in
Example 8
[0238] Tissue Cross-Reactivity Profile of mAb01-01
[0239] To determine the binding characteristics of mAB01-01 to human CLL and healthy tissue in immunohistochemistry (IHC) experiments, immunostaining was performed on sections of spleen tissue expressing the IGLV3-21.sup.R110-BCR and of healthy spleen, skin, kidney, heart, and brain tissue.
[0240] Prior to IHC, the tissue sections were deparaffinized and hydrated. To unmask the antigens, microwave treatment with citrate buffer pH 6.0 (9 ml citric acid (0.1M) and 41 ml sodium citrate (0.1 M)) was performed. For this, the sections were boiled in the bubbling citrate buffer for 15 min, after which they chilled at room temperature for 30 min and then they were rinsed in PBS 3×5 min. For IHC, the slides were incubated for 2 hr with the first antibody at a dilution of 1:200 at RT in a humidity chamber. As a control, sections of all tissues were incubated under identical conditions without the first antibody. Thereafter, the slides were washed in PBS 3×5 min. An anti-IgG antibody conjugated with horseradish peroxidase (HRP) (Goat Anti-Mouse IgG(H+L)-HRP, Southern Biotech, Cat. No. 1036-05) as a secondary antibody was incubated for 1 hour at a dilution of 1:10000 at RT in a humidified chamber. Subsequently, it was washed with PBS for 10 min and a DAB substrate kit (#34065, Thermo Fisher) was used to detect the activity of the HRP. DAB (3,3′-Diaminobenzidine tetrahydrochloride) substrate was incubated for 15 min. Fluoromount-1 was used as the capping agent. The evaluation was done after 30 minutes and showed an insoluble, brown colored reaction product at the sites where HRP conjugated anti-IgG antibody bound to the tissue.
[0241] As shown in
Example 9
[0242] Test of Anti-IGLV3-21.sup.R110-BCR Antibodies in a Patient Derived Xenograft Model
[0243] To determine the efficacy of the anti-IGLV3-21.sup.R110 antibodies a patient derived xenograft model was chosen. For a Dose finding experiment 4 groups with 4 NOD-scid IL2rg null (NSG)-mice (Jackson ImmunoResearch, prepared as described in Qi J et al.: An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies, Methods 2019, 154:70-76) were used: [0244] Group A: control group without antibody treatment [0245] Group B: dose 0.3 mg/kg body weight [0246] Group C: dose 5 mg/kg body weight [0247] Group D: dose 10 mg/kg body weight
[0248] PMBS from an IGLV3-21.sup.R110-BCR patient were thawed and resuspended in PBS. T cells where separated by using Miltenyi CD3 Beads (Miltenyi Biotec) following the instruction for use provided by the manufacturer. T cells were cultured and expanded for 7 days using CD3/CD28 dynabeads (Dynabeads™ Human T-Activator CD3/CD28 for T Cell Expansion and Activation, Cat. No. 11161D, GIBCO) as described before (Qi J et al. Methods, 2019 s.a.).
[0249] After 7 days, the activated T cells and PBMCs (20×10.sup.6 CLL PBMCs and 5×10.sup.5 T cells per mouse) were injected i.v. into NSG mice. For treatment mAb01-01 was given in different dosages i.p., twice a week for total 3 weeks, starting at week 2 post engraftment. Mice where pre-conditioned at the beginning of every week with 250 μl human serum. The mice were sacrificed after 3 weeks of treatment. For analysis the spleen was isolated and analyzed for the existence of human IGLV3-21.sup.R110 positive CLL B cells by flow cytometry using the mAb01-01 and antibodies against human CD45, CD5S, and CD19 (CD5 IgG1 UCHT2, BioLegend; CD19 IgG1 HIB19, BD Biosciences; CD45 (human) IgG1 H130 Invitrogen). For flow cytometry, cells were collected by centrifugation and resuspended in ice-cold 0.1% (w/v) BSA in PBS (flow cytometrybuffer). 100 μL containing 5×10.sup.5 cells were distributed into a V-bottom 96-well plate (Corning). The cells were first blocked with 5% (v/v) goat serum (Jackson ImmunoResearch) for 30 min on ice and then incubated with the indicated antibodies as recommended by the manufacturer. The cells were incubated for 30 min on ice in dark. Then the cells were washed twice with ice-cold flow cytometry buffer, resuspended in 200 μLflow cytometry buffer and analyzed using FACSCanto (BD Biosciences).
[0250] As shown in