Anti-BTN3A antibodies and their use in treating cancer or infectious disorders
11987631 · 2024-05-21
Assignee
- INSERM (INSTITUT NATIONAL DE LA SANT? ET DE LA RECHERCHE M?DICALE) (Paris, FR)
- UNIVERSIT? AIX MARSEILLE (Marseilles, FR)
- INSTITUT JEAN PAOLI & IRENE CALMETTE (Marseilles, FR)
- CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE—CNRS (Paris, FR)
Inventors
- Alemseged Truneh (Sudbury, MA, US)
- Daniel Olive (Marseilles, FR)
- Christine Pasero (Marseilles, FR)
- Aude De Gassart (Marseilles, FR)
Cpc classification
C07K2317/33
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
A61K2239/38
HUMAN NECESSITIES
C07K2317/92
CHEMISTRY; METALLURGY
A61K2039/545
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K39/4611
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
C07K2317/94
CHEMISTRY; METALLURGY
A61K2039/507
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61K9/19
HUMAN NECESSITIES
A61K2239/59
HUMAN NECESSITIES
International classification
A61K9/19
HUMAN NECESSITIES
A61K39/00
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to humanized antibodies that specifically bind to human BTN3A and their use in treating cancer and infectious disorders.
Claims
1. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an anti-BTN3A antibody comprising a variable heavy chain polypeptide VH of SEQ ID NO:1 and a variable light chain polypeptide VL of SEQ ID NO:2 or SEQ ID NO: 3.
2. The method of claim 1, wherein said anti-BTN3A antibody is administered in combination with an anti-PD1 or anti-PD-L1 antibody.
3. The method of claim 1, wherein said anti-BTN3A antibody binds to human BTN3A1 polypeptide with a KD of 5 nM or less as measured by surface plasmon resonance.
4. The method of claim 1, wherein said anti-BTN3A antibody induces the activation of V?9V?2-T cells in co-culture with BTN3A expressing cells, with an EC.sub.50 of 1 ?g/ml or below, as measured in a degranulation assay.
5. The method of claim 1, wherein said anti-BTN3A antibody is administered in combination with IL-2 or IL-15 or their derivatives.
6. The method of claim 1, wherein said anti-BTN3A antibody comprises a mutant or chemically modified IgG1 constant region, wherein said mutant or chemically modified IgG1 constant region confers no or decreased binding to Fc? receptors when compared to a corresponding antibody with wild type IgG1 isotype constant region.
7. The method of claim 6, wherein said mutant IgG1 constant region is IgG1 triple mutant L247F, L248E and P350S.
8. The method of claim 1, wherein said anti-BTN3A antibody comprises a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.
9. The method of claim 1, wherein said anti-BTN3A antibody is administered in combination with an anti-PD1 antibody selected from the group consisting of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, and atezolizumab.
10. The method of claim 1, wherein said anti-BTN3A antibody is administered in combination with pembrolizumab.
11. The method of claim 1, wherein said cancer is a non-hematological cancer.
12. The method of claim 1, wherein said method comprises administering a therapeutically efficient amount of an anti-BTN3A antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6, and wherein said cancer is a non-hematological cancer.
13. The method of claim 1, wherein said method comprises administering a therapeutically efficient amount of an anti-BTN3A antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6, in combination with an anti-PD1 antibody, and wherein said cancer is a non-hematological cancer.
14. The method of claim 1, wherein said method comprises administering a therapeutically efficient amount of an anti-BTN3A antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6, in combination with pembrolizumab, and wherein said cancer is a non-hematological cancer.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
(4)
(5) A. Cynomolgus whole blood from 3 animals was treated with red blood cells lysis buffer. After extensive washes, cells were plated at 1.5 M/mL in medium containing 200 IU/mL rHuIL-2 and mAb1 (10 ?g/mL). Percentage of V?9+ T cells was assessed by flow cytometry at day 0, 3, 6, 8 and 10 using a specific antibody. Graph shows the kinetic of V?9+ T cell percentage among live cells. Each curve represents an individual animal.
(6) B. After 10 days expansion, cells from each animal were cocultured for 4 hrs with Daudi, K562 or Raji used as target cells (ratio E:T 1:1) in presence of culture medium, mAb1 or isotype control (10 ?g/mL) and analysed for degranulation (CD107a/b) by flow cytometry.
(7)
EXAMPLES
(8) Selection of Humanized Variants
(9) 1. Description of Humanization Strategies
(10) a. Design of Composite Human Antibody? Variable Region Sequences
(11) Structural models of the murine 7.2 and 20.1 antibody V regions were produced using Swiss PDB and analyzed in order to identify important constraining amino acids in the V regions that were likely to be essential for the binding properties of the antibodies. Most residues contained within the CDRs (using both Kabat and Chothia definitions) together with a number of framework residues were considered to be important. From the above analysis, Composite Human sequences of 7.2 and 20.1 antibodies have been created.
(12) b. CD4+ T Cell Epitope Avoidance
(13) Based upon the structural analysis, a large preliminary set of sequence segments that could be used to create 7.2 and 20.1 humanized variants were selected and analyzed using iTope? technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008), and using the TCED? of known antibody sequence-related T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II or that scored significant hits against the TCED? were discarded. This resulted in a reduced set of segments, and combinations of these were again analyzed, as above, to ensure that the junctions between segments did not contain potential T cell epitopes. Selected sequence segments were assembled into complete V region sequences predicted to be devoid of significant T cell epitopes. Several heavy chains and light chains sequences were then chosen for gene synthesis and expression in mammalian cells for mAbs 7.2 and 20.1.
(14) 2. Generation of Humanized Variants and Preliminary Characterization
(15) a. Construction of Humanized Variants Plasmids
(16) 7.2 and 20.1 humanized variants were synthesized with flanking restriction enzyme sites for cloning into an expression vector system for human IgG4 (S241P, L248E) heavy and kappa light chains. All constructs were confirmed by sequencing.
(17) b. Expression of Antibodies
(18) Chimeric 7.2 and 20.1 (VH0/V?0), two control combinations (VH0/V?1, VH1/V?0) and combinations of humanized heavy and light chains were transiently transfected into FreeStyle? CHO-S cells (ThermoFisher, Loughborough, UK) using a MaxCyte STX? electroporation system (MaxCyte Inc., Gaithersburg, USA) from corresponding endotoxin-free DNA. Transfections were undertaken for each antibody using OC-400 processing assemblies. Following cell recovery, cells were diluted to 3?10.sup.6 cells/mL into CD Opti-CHO medium (ThermoFisher, Loughborough, UK) containing 8 mM L-Glutamine (ThermoFisher, Loughborough, UK) and 1?Hypoxanthine-Thymidine (ThermoFisher, Loughborough, UK). 24 hours post-transfection, the culture temperature was reduced to 32? C. and 1 mM sodium butyrate (Sigma, Dorset, UK) was added. Cultures were fed daily by the addition of 3.6% (of the starting volume) feed (2.5% CHO CD Efficient Feed A (ThermoFisher, Loughborough, UK), 0.5% Yeastolate (BD Biosciences, Oxford, UK), 0.25 mM Glutamax (ThermoFisher, Loughborough, UK) and 2 g/L Glucose (Sigma, Dorset, UK)). IgG supernatant titers were monitored by IgG ELISA and transfections were cultured for up to 14 days prior to harvesting supernatants.
(19) c. Preliminary Affinity Measurement: Single Cycle Kinetic Analysis of Humanized Variants Binding to BTN3A
(20) In order to assess the binding of all 7.2 and 20.1 Composite Human Antibody? variants and to select antibodies with the highest affinity to BTN3A, single cycle kinetic analysis was performed on supernatants from transfected cell culture using a Biacore T200 (serial no. 1909913) running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).
(21) Antibodies were diluted in 2% BSA/PBS to a final concentration of 2 ?g/ml based on concentrations obtained from the supernatant titered by ELISA. At the start of each cycle, antibodies were loaded onto Fc2, Fc3 and Fc4 of the Protein A chip (GE Healthcare, Little Chalfont, UK). IgGs were captured at a flow rate of 10 ?l/min to give an immobilization level (RL) of ?146.5 RU, the theoretical value to obtain RMax of ?50 RU. The surface was then allowed to stabilize. Single cycle kinetic data was obtained with BTN3A1-His as the analyte (Sino Biological Cat. No. 15973-H08H) at a flow rate of 60 ?l/min to minimize any potential mass transfer effects, as well as using HBS-P+ (GE Healthcare, Little Chalfont, UK) as running buffer. Multiple repeats with the chimeric antibody were performed to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 (no antibody) was subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. A three point, four-fold dilution range from 1.56 nM to 25 nM BTN3A1 without regeneration between each concentration was used. The association phase for the three injections of increasing concentrations of BTN3A1 was monitored for 240 seconds each time and a single dissociation phase was measured for 2000 seconds following the last injection of BTN3A1. Regeneration of the Protein A surface was conducted using two injections of 10 mM glycine-HCL pH 1.5 followed by a stabilization period of 240 seconds.
(22) The signal from each antibody blank run (no CD277) was subtracted to correct for differences in surface stability. Single cycle kinetics demonstrated that all humanized variants bound to BTN3A.
(23) d. Purification of Antibodies
(24) Based on the affinities calculated by Biacore, as well as the iTope? score and percentage of humanness of each humanized variant, 7.2 and 20.1 humanized variants with the best affinities and best iTope? scores were selected for further analysis.
(25) The selected humanized variants together with their chimeric version and the most conservatively humanized variant (VH1/V?1) were subjected to purification for further assay testing. Antibodies were purified from cell culture supernatants on Protein A sepharose columns followed by Size Exclusion Chromatography (SEC) (GE Healthcare, Little Chalfont, UK) using 10 mM sodium acetate, 100 mM NaCl, pH 5.5 as mobile phase and final formulation buffer. Samples were quantified by OD.sub.280nm using an extinction coefficient (Ec(0.1%)) based on the predicted amino acid sequence.
(26) Antibodies were analyzed using SDS-PAGE by loading 2 ?g of each antibody on the gel and bands corresponding to the profile of a typical antibody were observed.
(27) e. Validation of Binding Properties: Competition ELISA Analysis Between Humanized and Chimeric 7.2 and 20.1 mAbs
(28) Purified variants were tested for their binding to recombinant BTN3A1-His (Sino Biological cat. no. 15973-H08H) while competing against the corresponding murine antibody. Chimeric (VH0/V?0) and irrelevant human IgG4 (S241P, L248E) were tested on each plate for comparison.
(29) BTN3A1 was diluted in 1?PBS to 0.5 ?g/ml and 100 ?l/well was coated overnight at 4? C. on a 96-well ELISA plate. The following day, the plate was washed 3? with 1?PBS/0.05% Tween (PBS-T) and blocked with 200 ?l of 2% milk/PBS for one hour at room temperature. In a dilution 96-well plate a fixed concentration of murine antibodies 7.2 or 20.1 (0.15 ?g/ml final concentration) was added in equal volume to a four-fold titration series of test antibody (starting from 80 ?g/ml (40 ?g/ml final concentration) diluted in blocking buffer). After washing the Nunc ELISA plate 3? with PBS-T, 100 ?l of murine/test antibody mix was added to the ELISA plate. After one hour incubation at room temperature, the plate was washed 3? with PBS-T and 100 ?l of anti-mouse Fc HRP-labelled secondary antibody (Sigma, Dorset, UK) diluted 1:1000 in blocking buffer was applied for one hour at room temperature to detect bound murine antibody. For color development, the plate was washed 3? with PBS-T following which 100 ?l of TMB substrate was added and incubated for five minutes at room temperature. The reaction was stopped with 100 ?l of 3.0 M hydrochloric acid and absorbance was read immediately using a Dynex plate reader at 450 nm.
(30) IC.sub.50 values were calculated for each variant and relative IC.sub.50 values were calculated by dividing the IC.sub.50 of the humanized variant by that of the chimeric antibody assayed on the same plate.
(31) 3. Selection of the Humanized Candidates
(32) a. Multi-Cycle Kinetic Analysis
(33) Based on the data generated from competition ELISA and thermal stability assessment, multi-cycle kinetic analysis was performed on most of the humanized 7.2 and 20.1 variants together with the VH0/V?0 chimeric antibody using a Biacore T200 (serial no. 1909913) instrument running Biacore T200 Evaluation Software V2.0.1 (Uppsala, Sweden).
(34) Purified antibodies were diluted to a concentration of 2 ?g/ml in 2% BSA/PBS. At the start of each cycle, each antibody was captured on the Protein A at a density (RL) of ?146.5 RU (theoretical value to obtain an RMax of ?50 RU). Following capture, the surface was allowed to stabilize before injection of the BTN3A1 antigen (Sino Biological cat. no. 15973-H08H). BTN3A1 was titrated in 0.1% BSA/HBS-P+ (running buffer) in a two-fold dilution range from 25 to 0.78 nM. The association phase was monitored for 400 seconds and the dissociation phase for 35 minutes (2100 seconds). Kinetic data was obtained using a flow rate of 50 ?l/min to minimize any potential mass transfer effects. Regeneration of the Protein A surface was conducted using two injections of 10 mM glycine-HCL pH 1.5 at the end of each cycle. Two blanks (no BTN3A1) and a repeat of a single concentration of the analyte were performed for each tested antibody to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 was subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to a reference surface. Additionally, blank runs were subtracted for each Fc to correct any antigen-independent signal variation, such as drift. Sensorgrams were fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (Constant RI=0 RU).
(35) b. Binding Assay by Flow Cytometry on Human PBMCs
(36) 7.2 and 20.1 humanized variants were characterized for their binding to human PBMCs, isolated from blood of healthy donors. PBMCs were isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs were then frozen and stored at ?80? C. or in liquid nitrogen until required.
(37) 100 ?l cells at 1?10.sup.6 cells/ml were transferred to each well of a fresh U-shaped bottom 96-well plate, then the plate was centrifuged and supernatant discarded.
(38) A serial dilution of the antibodies, 0.001 ?g/ml to 150 ?g/ml was prepared in PBS 2 mM EDTA. Human PBMCs were resuspended in 50 ?l of the diluted test antibody titration series prepared.
(39) After incubation for 30 minutes at 4? C. in the dark, the plate was centrifuged and washed twice with 150 ?l/well of PBS 2 mM EDTA following which the wells were resuspended in 50 ?l of a mix composed of goat anti-human antibody (PE labelled) diluted 1/100 and Live/dead neat IR diluted 1/500 in PBS 2 mM EDTA.
(40) After incubation for 15 minutes at 4? C. in the dark, the plate was centrifuged and washed once with 150 ?l/well PBS 2 mM EDTA following which the wells were resuspended in 200 ?l PBS 2 mM EDTA. Cells were analyzed on a BD LSR Fortessa Cytometer. Data was analyzed using a FlowJo software (Version 10, FlowJo, LLC, Ashland, USA) (Data not shown).
(41) Same protocol was performed on cynomolgus PBMCs and on Daudi Burkitt's lymphoma cell line.
(42) c. In Vitro Functional Efficacy: ??-T Cell Degranulation Assay
(43) The assay consists of measuring activating or inhibitory effect of 7.2 and 20.1 humanized variants and their chimeric versions on ??-T cell degranulation against Daudi Burkitt's lymphoma cell line (Harly et al., 2012). ??-T cells were expanded from PBMCs of healthy donors by culturing with zoledronic acid (1 ?M) and IL2 (200 Ui/ml) for 11-13 days. IL2 is added at day 5, day 8 and every 2 days thereafter. The percentage of ??-T cells was determined at the initiation of culture and assessed for the time of culture by flow cytometry until it reached at least 80%. Frozen or fresh ??-T cells were then used in degranulation assays against Daudi cell line (E:T ratio of 1:1), whereby the cells are co-cultured for 4 hours at 37? C. in presence of 10 ?g/ml of the 7.2 and 20.1 humanized variants and their chimeric versions. Activation by PMA (20 ng/ml) plus lonomycin (1 ?g/ml) served as positive control for ??-T cell degranulation, and medium alone as negative control. At the end of 4 hour co-incubation, cells were analyzed by flow cytometry to evaluate the percentage of ??-T cells positive for CD107a (LAMP-1, lysosomal-associated membrane protein-1)+CD107b (LAMP-2). CD107 is mobilized to the cell surface following activation-induced granule exocytosis, thus measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells. The results did not show any significant variations among the tested candidates, which showed similar activating effect in the degranulation assay as the chimeric 7.2 or 20.1 antibody.
(44) The same protocol was performed using AML blasts isolated from patients as target cells, in place of Daudi cells.
(45) d. Thermostability Analysis
(46) In order to assess the thermostability of the selected 7.2 and 20.1 Composite Human Antibody? variants, melting temperatures (the temperature at which 50% of a protein domain is unfolded) were determined using a fluorescence-based thermal shift assay.
(47) All purified humanized antibodies, and the chimeric (VH0/V?0) antibodies, were diluted to a final concentration of 0.1 mg/ml in formulation buffer (10 mM sodium acetate, 100 mM NaCl, pH 5.5) containing SYPRO? Orange (ThermoFisher, Loughborough, UK) at 1 in 1000 dilution and subjected to a temperature gradient from 25? C. to 99? C. on a StepOnePlus real-time PCR system (ThermoFisher, Loughborough, UK) over a period of 56 minutes. 10 mM sodium acetate, 100 mM NaCl, pH 5.5 was used as a negative control. The melting curves were analyzed using protein thermostability software (version 1.2).
(48) e. Selection of Humanized Candidates
(49) Based on all the results obtained for the experiments described above, 3 variants out of 35 (15 humanized variants generated for 7.2; 20 humanized variants generated for 20.1) were selected for further characterization: 7.2 (VH2/Vk1), 7.2 (VH2/Vk2) and 20.1 (VH3/Vk1).
(50) The results of the different experiments described above for the mAbs 7.2 and 20.1 are reported in the Table 4 and Table 5 for the 3 variants and their chimeric versions.
(51) TABLE-US-00004 TABLE 4 The selected humanized candidates have the same potency as the murine parent antibodies 7.2 7.2 7.2 20.1 20.1 Candidate VH2/Vk1 VH2/Vk2 VH0/Vk0 VH3/Vk1 VH0/Vk0 Biacore 2.53 2.43 1.76 3.19 2.34 affinity Multi-Cycle Kinetics (?10.sup.?10) (K.sub.D, M) Binding on 3.86 5.94 3.6 3.38 3.02 human PBMC (EC.sub.50, ?g/mL) Binding 12.0 9.85 7.75 5.74 4.06 Cyno PBMC (EC.sub.50, ?g/mL) Binding to 2.02 2.01 1.44 1.59 1.19 lymphoma (Daudi) (EC.sub.50, ?g/mL) Functional 0.03 0.03 0.02 0.02 0.02 assay (Daudi) (??T cell-based, EC.sub.50, ?g/mL) Functional 0.21 0.19 0.12 0.15 0.05 assay (AML sensitive) (??T cell-based, EC.sub.50, ?g/mL) Functional 0.73 0.64 0.42 0.32 0.18 assay (AML resistant) (??T cell-based, EC.sub.50, ?g/mL)
(52) TABLE-US-00005 TABLE 5 The humanized selected candidates mAb7.2 with VH2 and either Vk1 or Vk2 have higher thermostability as compared to murine candidate Candidate 7.2 VH2/Vk1 7.2 VH2/Vk2 7.2 VH0/Vk0 Thermostability 77.1 77.3 72.5 (T.sub.m2 mean) (? C.)
(53) The humanization process lead to the generation of multiple 7.2 and 20.1 variants with predicted reduced immunogenicity.
(54) The selected set of the three variants (7.2 VH2/VK1, 7.2 VH2/VK2 and 20.1 VH3/VK1) showed equivalent properties as their chimeric version in terms of affinity, binding and efficacy in functional assays: the modifications made in the variants sequences to reduce immunogenicity did not alter the antibodies functions.
(55) Surprisingly, the thermostability of the selected humanized variants 7.2 VH2/VK1, 7.2 VH2/VK2 was improved compared to the chimeric antibodies, and such improved thermostability was unexpected in this process of humanization.
(56) Constant Region of the Antibody: Comparison of Silent Fc Fragments
(57) Several Fc portions were tested to silence or reduce the effector function of the antibodies. The binding of these Fc fragments to the different Fc? receptors was assessed using Biacore; their binding on C1q complex was assessed by ELISA assay.
(58) 1. Binding of the Engineered Fc Portion to the Different Fc? Receptors Using Biacore
(59) The ability of different isotypes (IgG1, IgG1 [N314A], IgG1 [L247F, L248E P350S], IgG2, IgG4 [S241P] and IgG4 [S241P L248E]) of the chimeric antibody 20.1 to bind to different Fc? receptors was determined using purified antibodies and single cycle Biacore analysis. The human Fc receptors, Fc?RI, Fc?RIIA (Arg167 polymorphism) and IIB, and Fc?RIIIA (Phe176 polymorphism) and IIIB were obtained from Sino Biological.
(60) Fc? receptors were diluted in HBS-P+ (GE Healthcare, Little Chalfont, UK) to a final concentration of either 0.5 or 1.0 ?g/ml. At the start of each cycle, Fc? receptors were loaded onto Fc2, Fc3 and Fc4 of an anti-His CM5 chip (GE Healthcare, Little Chalfont, UK). Fc? receptors were captured at a flow rate of 5 ?l/min to give an immobilization level of between 30 and 180 RU depending on the molecular weight of the Fc? receptor. The surface was then allowed to stabilize. Single-cycle kinetics data was obtained using the chimeric antibodies as the analyte at a flow rate of 30 ?l/min to minimize any potential mass transfer effects. The signal from the reference channel Fc1 (no antibody) was subtracted from that of Fc2, Fc3 and Fc4 to correct for differences in non-specific binding to the reference surface. A five point, three-fold dilution range was used for each chimeric antibody with this concentration range varying for each individual Fc? receptor due to the expected differences in affinity. The signal from each blank run (no antibody) was subtracted to correct for differences in surface stability. The association phase for each of the five injections of increasing concentrations of chimeric antibody was monitored for between 25 and 180 seconds (depending on the Fc? receptor ligand) and a single dissociation phase was measured for between 25 and 300 seconds following the last injection of antibody. Regeneration of the anti-His surface was conducted using two injections of 10 mM glycine-HCL pH 1.5 for 15 seconds each at 30 ?l/min followed by a stabilization period of 180 seconds.
(61) Single-cycle kinetic constants were determined where possible using the standard 1:1 analysis model. For strong interactions it was generally more suitable to determine affinity via kinetic experiments. However, for several Fc? receptors, the interaction is very weak and in this scenario, the data was analyzed using steady state affinity analysis (which is particularly suited to measurement of weak to moderate interactions). Sensorgrams for the interactions of the Fc? receptors with the chimeric antibodies were obtained (data not shown).
(62) As expected, the high affinity Fc?RI receptor bound with good affinity to unmodified IgG1 and IgG4 (S241P). The modified IgG1 isotypes, together with IgG2 and IgG4 (S241P, L248E) failed to bind to Fc?RI. The remaining Fc? receptors showed much lower affinity interactions for the different chimeric antibodies compared to the Fc?RI. As expected, the unmodified IgG1 showed the strongest binding to all four of the lower affinity receptors, whereas the modified versions of IgG1 showed significantly reduced binding to these receptors. IgG2 and IgG4 (S241P) demonstrated some binding to Fc?RIIA and B but only marginal binding to Fc?RIIIA and B.
(63) 2. Binding of the Engineered Fc Portion to the C1q Complex by ELISA Assay
(64) The chimeric antibody 20.1 was tested as different IgG isotypes for binding to the C1q complex to determine their ability to activate the complement system.
(65) In a U-bottomed 96-well plate, a 2.5-fold dilution series (from 10 ?g/ml to 0.04 ?g/ml) of purified chimeric 20.1 in different isotypes was prepared in 2% BSA/DPBS. Nunc Immuno MaxiSorp 96 well flat bottom microtitre plates (ThermoFisher Scientific, Loughborough, UK) were pre-coated with 100 ?l/well of this titration series and incubated overnight at 4? C. The following day the plate was washed twice with PBST and blocked for one hour at room temperature with 2% BSA/DPBS before washing five times with PBST. Purified complement protein C1q (Pathway Diagnostics Ltd, Dorking, UK), diluted to 5 ?g/ml in 2% BSA/PBS, was added to the plate (100 ?l/well) and incubated for one hour at room temperature. After washing five times with PBST, the binding of C1q complex was detected with an anti-C1q-HRP (Abcam, Cambridge, UK) (100 ?l/well, diluted 1 in 100 in 2% BSA/DPBS) for one hour at room temperature. After washing five times with PBST, binding was detected with TMB substrate (ThermoFisher Scientific, Loughborough, UK) following which the reaction was stopped with 3 M HCl, absorbance read at 450 nm on a Dynex Technologies MRX TC II plate reader and the binding curves plotted.
(66) As expected, only the unmodified IgG1 isotype showed good binding to C1q with other isotypes showing minimal to no binding (data not shown).
(67) 3. Selection of the Engineered Fc Fragments
(68) The relative binding of all tested isotypes on Fc? receptors and C1q complex are described in Table 6.
(69) TABLE-US-00006 TABLE 6 Relative binding of all isotypes on Fc? receptors and C1q complex Isotype Fc?RI Fc?RIIa Fc?RIIb Fc?RIIIa Fc?RIIIb C1q IgG1 IgG1 WT ++++ ++ ++ ++ ++ ++++ IgG1 N314A ++ ? ? ? ? ? IgG1 L247F, L248E, ? + + +/? +/? P350S IgG2 IgG2 WT ? ++ + +/? +/? +/? IgG4 IgG4 S241P ++++ ++ ++ +/? +/? ? IgG4 S241P, L248E ? + ++ ? +/? ?
(70) The two engineered IgG1 L247F, L248E, P350S and IgG4 S241P, L248E Fc fragments were the only one to show a total loss of binding on Fc?1 receptor and C1q complex.
(71) Based on the results obtained, the two engineered IgG1 L247F, L248E, P350S and IgG4 S241P, L248E isotypes were selected for further characterization.
(72) Generation of 6 Humanized Antibodies
(73) The 3 selected humanized variants were cloned to be fused with the two selected engineered Fc fragments, leading to the generation of 6 different candidates: mAb 1 to mAb 6.
(74) The Examples mAb1 to mAb6 as described in Table 1 can be produced using conventional antibody recombinant production and purification processes.
(75) For example, the coding sequences have been cloned into a production vector for recombinant expression in mammalian production cell line.
(76) The following Tables 7 and 8 provides detailed amino acid and nucleotide sequences useful for practicing the invention, and in particular for producing the nucleic acids, expression vectors and humanized antibodies derived from the murine 7.2 of the present disclosure.
(77) TABLE-US-00007 TABLE 7 Brief description of useful amino acid and nucleotide sequences for practicing the invention SEQ ID NO: Type Description of the sequence 1 aa Humanized heavy chain variable region VH2 of mAb 7.2 2 aa Humanized light chain variable region V?1 of mAb 7.2 3 aa Humanized light chain variable region V?2 of mAb 7.2 4 aa Full length heavy chain of mAbs 1 and 2 (VH2 7.2 silent IgG1) 5 aa Full length heavy chain of mAbs 4 and 5 (VH2 7.2 silent IgG4) 6 aa Full length light chain of mAbs 1 and 4 (Vk1 7.2) 7 aa Full length light chain of mAbs 2 and 5 (Vk2 7.2) 8 nt Full length heavy chain of mAbs 1 and 2 (VH2 7.2 silent IgG1) 9 nt Full length heavy chain of mAbs 4 and 5 (VH2 7.2 silent IgG4) 10 nt Full length light chain of mAbs 1 and 4 (Vk1 7.2) 11 nt Full length light chain of mAbs 2 and 5 (Vk2 7.2) 12 aa HCDR1 of mAb 7.2, 1, 2, 4 and 5 13 aa HCDR2 of mAb 7.2, 1, 2, 4 and 5 14 aa HCDR3 of mAb 7.2, 1, 2, 4 and 5 15 aa LCDR1 of mAb 7.2, 1, 2, 4 and 5 16 aa LCDR2 of mAb 7.2, 1, 2, 4 and 5 17 aa LCDR3 of mAb 7.2, 1, 2, 4 and 5 18 aa Human BTN3A1 19 aa Human BTN3A2 20 aa Human BTN3A3 21 aa Cynomolgus macaque (m. fascicularis) BTN3A1 ectodomain used for recombinant protein production 22 aa Cynomolgus macaque (m. fascicularis) BTN3A2 ectodomain used for recombinant protein production 23 aa Cynomolgus macaque (m. fascicularis) BTN3A3 ectodomain used for recombinant protein production
(78) TABLE-US-00008 TABLE8 Briefdescriptionofusefulaminoacidandnucleotide sequencesforpracticingtheinvention SEQID NO: Describestheaminoacidornucleotidesequencebelow: 1 QVQLVQSGAEVKKPGASVKLSCKASGYIFTRYYMYWVKQRPGQGLEWIGEI NPNNGGTKFNEKFKNRATLTVDKSISTAYMELSRLRSDDTAVYYCSREDDY DGTPFAMDYWGQGTLVTVSS 2 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKAS NLHTGVPSRFTGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKL EIK 3 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKAS NLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKL EIK 4 QVQLVQSGAEVKKPGASVKLSCKASGYIFTRYYMYWVKQRPGQGLEWIGEI NPNNGGTKFNEKFKNRATLTVDKSISTAYMELSRLRSDDTAVYYCSREDDY DGTPFAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY1 CNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDT LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 5 QVQLVQSGAEVKKPGASVKLSCKASGYIFTRYYMYWVKQRPGQGLEWIGEI NPNNGGTKFNEKFKNRATLTVDKSISTAYMELSRLRSDDTAVYYCSREDDY DGTPFAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKD YFPEPVIVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVIVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG 6 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKAS NLHTGVPSRFTGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 7 DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKAS NLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGQTYPYTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC 8 CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCT TCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACATCTTCACCAGATACTA TATGTATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA GAGATTAATCCTAACAATGGTGGTACTAAGTTCAATGAGAAGTTCAAGAA CAGGGCCACACTGACTGTAGACAAATCCATCAGCACAGCATACATGGAG CTCAGCAGGCTGAGATCTGACGACACGGCGGTCTATTATTGTTCAAGAG AGGATGATTACGACGGGACCCCCTTTGCTATGGACTACTGGGGTCAAGG AACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTC CCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACT CACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGGGGACCGTCA GTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGA CCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTG AGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAA GACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAG CGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAA GTGCAAGGTCTCCAACAAAGCCCTCCCAGCCTCCATCGAGAAAACCATC TCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGAAGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCC GACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGT GGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCA CAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTTGA 9 CAGGTCCAACTGGTGCAGTCTGGGGCTGAAGTGAAGAAGCCTGGGGCT TCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACATCTTCACCAGATACTA TATGTATTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGA GAGATTAATCCTAACAATGGTGGTACTAAGTTCAATGAGAAGTTCAAGAA CAGGGCCACACTGACTGTAGACAAATCCATCAGCACAGCATACATGGAG CTCAGCAGGCTGAGATCTGACGACACGGCGGTCTATTATTGTTCAAGAG AGGATGATTACGACGGGACCCCCTTTGCTATGGACTACTGGGGTCAAGG AACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTC CCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTG GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCA GCAGCTTGGGCACGAAGACCTACACCTGCAATGTAGATCACAAGCCCAG CAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGC CCACCATGCCCAGCACCTGAGTTCGAGGGGGGACCATCAGTCTTCCTGT TCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGT CACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTT CAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCAC CGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGT CTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCC AAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAG GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC TTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG GAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGG GGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTA CACACAGAAGAGCCTCTCCCTGTCTCTGGGTTGA 10 GACATCCAGATGACCCAGTCTCCATCCAGTCTGTCTGCATCCGTAGGAG ACAGAGTCACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTA TCTTGGTACCAGCAGAAACCAGGAAAAGCCCCTAAACTCTTGATCTATAA GGCTTCCAACTTGCACACAGGCGTCCCATCAAGATTTACTGGCAGTGGA TCTGGAACAGATTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACAT TGCCACTTACTACTGTCAACAGGGTCAAACTTATCCATACACGTTCGGAC AGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTT CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACC CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTTAG 11 GACATCCAGATGACCCAGTCTCCATCCAGTCTGTCTGCATCCGTAGGAG ACAGAGTCACCATCACTTGCCATGCCAGTCAGAACATTAATGTTTGGTTA TCTTGGTACCAGCAGAAACCAGGAAAAGCCCCTAAACTCTTGATCTATAA GGCTTCCAACTTGCACACAGGCGTCCCATCAAGATTTAGTGGCAGTGGA TCTGGAACAGATTTCACATTCACCATCAGCAGCCTGCAGCCTGAAGACAT TGCCACTTACTACTGTCAACAGGGTCAAACTTATCCATACACGTTCGGAC AGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTT CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTG TGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAA GGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGA GCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCT GAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGICACC CATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG TGTTAG 12 RYYMY 13 EINPNNGGTKFNEKFKN 14 EDDYDGTPFAMDY 15 HASQNINVWLS 16 KASNLHT 17 QQGQTYPYT 18 MKMASFLAFLLLNFRVCLLLLQLLMPHSAQFSVLGPSGPILAMVGEDADLPC HLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRD GITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHVDV KGYKDGGIHLECRSTGWYPQPQIQWSNNKGENIPTVEAPVVADGVGLYAV AASVIMRGSSGEGVSCTIRSSLLGLEKTASISIADPFFRSAQRWIAALAGTLP VLLLLLGGAGYFLWQQQEEKKTQFRKKKREQELREMAWSTMKQEQSTRVK LLEELRWRSIQYASRGERHSAYNEWKKALFKPADVILDPKTANPILLVSEDQ RSVQRAKEPQDLPDNPERFNWHYCVLGCESFISGRHYWEVEVGDRKEWHI GVCSKNVQRKGWVKMTPENGFWTMGLTDGNKYRTLTEPRTNLKLPKPPKK VGVFLDYETGDISFYNAVDGSHIHTFLDVSFSEALYPVFRILTLEPTALTICPA 19 MKMASSLAFLLLNFHVSLLLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPC HLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRD GITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSNLHVEV KGYEDGGIHLECRSTGWYPQPQIQWSNAKGENIPAVEAPVVADGVGLYEV AASVIMRGGSGEGVSCIIRNSLLGLEKTASISIADPFFRSAQPWIAALAGTLPI LLLLLAGASYFLWRQQKEITALSSEIESEQEMKEMGYAATEREISLRESLQEE LKRKKIQYLTRGEESSSDTNKSA 20 MKMASSLAFLLLNFHVSLFLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPC HLFPTMSAETMELRWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRD GITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIEVK GYEDGGIHLECRSTGWYPQPQIKVVSDTKGENIPAVEAPVVADGVGLYAVAA SVIMRGSSGGGVSCIIRNSLLGLEKTASISIADPFFRSAQPWIAALAGTLPISLL LLAGASYFLWRQQKEKIALSRETEREREMKEMGYAATEQEISLREKLQEELK WRKIQYMARGEKSLAYHEWKMALFKPADVILDPDTANAILLVSEDQRSVQR AEEPRDLPDNPERFEWRYCVLGCENFTSGRHYWEVEVGDRKEWHIGVCS KNVERKKGWVKMTPENGYVVTMGLTDGNKYRALTEPRTNLKLPEPPRKVGI FLDYETGEISFYNATDGSHIYTFPHASFSEPLYPVFRILTLEPTALTICPIPKEV ESSPDPDLVPDHSLETPLTPGLANESGEPQAEVTSLLLPAHPGAEVSPSATT NQNHKLQARTEALY 21 MGSSLAFLLLSFHVCVLLLQLLMPHSAQFAVVGPPGPILAMVGEDADLPCHL FPTMSAETMELRWVSSNLRQVVNVYADGKEVEDRQSAAYRGRTSILRDGIT AGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIDVKGY EDGGIHLECRSTGWYPQPQIRWSNDKGENIPAVEAPVFVDGVGLYAVAASV ILRGSSGEGVSCTIRSSLLGLEKTTSISIAGHHHHHH 22 MGSSLAFLLLNFHVSFFLVQLLTPCSAQFSVLGPSGPILAMVGEDADLPCHL FPTMSAETMELRWVSSSLRQVVNVYADGKEVEDRQSAPYRGRTSILRDDIA AGKAALRIHNVTASDSGKYLCYFQDADFYEKALVELKVAALGSNLHVEVKGY EDGGIHLECRSTGWYPQPKIQWSNAKGQNIPAVEAPVVADGVGLYAVAASV IMRGGSGESVSCIIRNSVLGLEKTASISIADHHHHHH 23 MANFLAFLLLNFRVCLLLVQLLTPCSAQFAVLGPHGPILAMVGEDVDLPCHL FPTMSAETMELRWVSSSLRQVVNVYSDGKEVEDRQSAPYRGRTSILRDGIT AGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHIEVKGY EDGGIHLECRSTGWYPQPQIQWSNTKGQHIPAVKAPVVADGVGLYAVAASV IMRGSSGEGVSCIIRNSLLGLEKTASISITDHHHHHH
Developability Properties of the 6 Humanized Variants
(79) 1. Generation of Cell Lines
(80) a. Expression Vector Construction
(81) The gene sequences encoding the heavy chains and light chains were cloned into the vector. The gene system was used to express the antibodies in CHO cells. Antibodies expression was under the control of the EF1 alpha promoter. The expression vectors bear unique genetic elements that shield the transgene from the silencing effects of surrounding chromatin (Girod et al., 2007). Transcription is maintained at a maximum level and is independent of the transgene integration site, resulting in stable and high-level protein expression.
(82) b. Cell Lines Development
(83) The CHO host cell line is derived from CHO-K1 CCL-61 cells from the American 124 Type Culture Collection (ATCC) and has been adapted to grow in suspension in the chemically defined BalanCD Growth A culture medium (Irvine Scientific). Cells were transfected by electroporation using the Neon transfection system (Invitrogen).
(84) c. Single-Cell Cloning Using ClonePix FL Device.
(85) The same medium (BalanCD Growth A, Irvine Scientific) was used as a basal medium for transfection, single-cell cloning, and production in order to keep the environment of the cells unchanged throughout the whole procedure. Following transfection of each vector, puromycin selection pressure was applied to generate the stable pools. Diluted cells were plated into semi-solid media (CloneMedia?; Molecular Devices) and plates were incubated at 37? C. with 5% CO.sub.2, in a humidified incubator. Expanded colonies were picked using ClonePix? FL Imager from Molecular Devices and transferred to 96-well plates, then expanded in first 24-well and then 6-well TC plates.
(86) d. Fed-Batch Performance Evaluation
(87) Growth and production performance of individual clones were evaluated in 125 ml shake flasks to select the best clones by the criteria of cell growth performance and productivity in a 10 day fed-batch process using Cell Boost7 A+7B feed (GE Healthcare, USA). Fed-batch cultures were initiated at cell concentrations of 0.3?10.sup.6 cells/ml.
(88) Results obtained for cell growth and productivity are summarized in Table 9.
(89) 2. Samples Preparation for Manufacturability Assessment
(90) Each candidate antibody was purified by protein A capture from clarified CHO cell supernatant pools: two pools were required for each variant to ensure sufficient material for testing. The protein concentration was determined for all post-capture samples by UV method. Sample recovery and yields were greater than 80% for the majority of samples and all variants showed similar yields. Each antibody was then buffer-exchanged into 25 mM Histidine, 125 mM NaCl, pH 6.0 using 30 kDa MWCO centrifugal filter units until the flow-through material reached the target pH for the formulation. During the exchange step, there were no indicators of protein precipitation or slowed flow during the exchange for any of the variants. After buffer exchange completion, the concentration of each variant sample was adjusted to 1.0 mg/mL with formulation buffer, and 10% PS-80 was added to a final concentration of 0.02% PS-80.
(91) 3. Thermal Stability Assessment
(92) Differential scanning fluorimetry analysis was performed to assess and compare the thermal stabilities of tested antibody variant. Each variant was analyzed in triplicate and the mean T.sub.onset, T.sub.agg and T.sub.m determined for each observed thermal transition (data not shown).
(93) No significant differences were observed between T.sub.onset and T.sub.m values obtained for all tested variants. The determined T.sub.m1 value for all antibodies was 61? C., and the values determined for T.sub.onset were 54 to 55? C. for all antibodies. The T.sub.agg values determined based on the plots for colloidal stability ranged from 71 to 78? C.
(94) Overall, all 4 selected variants demonstrate comparable thermal stability: variations observed do not lead to significant changes in thermal stability between tested antibodies.
(95) 4. Forced Degradation Studies
(96) a. Agitation
(97) Samples for each variant were subjected to agitation stress on an orbital shaker set to 500 rpm at room temperature. One sample for each variant was agitated for 24 and another sample for 48 hours. One vial of each variant was stored at room temperature for up to 48 hours as a control. No changes in appearance were observed for agitated samples compared to controls: all samples were observed to be clear, colorless and free of visible particulates (data not shown). In addition, there was no significant change in total protein content as determined by UV method (data not shown).
(98) The effects of agitation stress on the stability of the panel of variants were evaluated by SEC, reduced CGE, non-reduced CGE, and icIEF methods (data not shown). No significant changes in the stability of the tested antibodies nor discernible trends over time in the accumulation of degradants were observed between the agitation control samples stored at room temperature and the agitation stress samples. The % main peak determined in SEC analysis was 99.2% for all control and agitation samples. In R-CGE analysis the % main peak was 98.5% for all control and agitation samples. NR-CGE analysis revealed no significant trends or changes in % main peak between the controls and stressed samples. In conclusion, no significant differences in stability were observed between all candidate variants.
(99) b. Freeze-Thaw Stress
(100) Three samples of each candidate were aliquoted into Eppendorf tubes, and subjected to freeze-thaw stress. Samples were stored at ?75?10? C. and then thawed at room temperature. One sample for each candidate was subjected to 3 freeze-thaw cycles; another to 6 freeze-thaw cycles; and a third sample to 10 cycles. All stressed samples were observed to be clear, colorless and free of visible particulates (data not shown), and there was no significant change in total protein content as determined by UV method (data not shown).
(101) The effects of freeze-thaw stress on the stability of the panel of variants were evaluated by SEC, reduced CGE, non-reduced CGE, and icIEF methods (data not shown). Freeze-thaw stress had no impact on the stability of the antibodies based on SEC, R-CGE and NR-CGE analysis.
(102) The icIEF analysis revealed noticeable changes in charge heterogeneity of tested antibodies. The concentration of basic variants decreased at successive F/T cycles, with the lowest concentration of basic variants at 10?F/T cycles as compared to general controls. The only exception was variant IgG1 7.2 VH2/VK1, for which the decrease in concentration of basic variants was at the same level for all tested F/T cycles (see Table 9). In variants mAb1, mAb2, and mAb3, after 10?F/T cycles, the basic species decreased by 1.0, 1.3, and 3.4%, respectively. The decrease in the concentration of the basic species is related to an increase in the % of main peak. In variants mAb4, mAb5 and mAb6 the decrease in basic species is 4.8, 2.8 and 4.7%, respectively. The change in variant mAb4 is mostly related to an increase of % main peak, while the changes in variant mAb5 and mAb6 are related to increases in both the % main peak and in the % acidic variant.
(103) Overall, it was determined that the highest resistance to changes from freeze-thaw stress was observed for variants mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2).
(104) c. Acidic pH Stress
(105) Samples for each candidate were subjected to acidic pH stress at room temperature: each sample was adjusted with HCl to pH 3.5 and kept at room temperature for 2 hours, 4 hours, and 24 hours after which samples were neutralized with 1 M Tris, pH 7.0. All samples were observed to be clear, colorless and free of visible particulates (data not shown), and there was no significant change in protein concentration as determined by UV method (data not shown).
(106) The effects of acidic pH stress on the panel of variants were evaluated by SEC, reduced CGE, non-reduced CGE, and icIEF methods (data not shown). No significant changes in the accumulation of degradants were detected over time for samples exposed to low pH as compared to 48 hours RT control by either R-CGE or NR-CGE analysis. In the charge heterogeneity analysis, the concentration of basic variants decreased in all samples subjected to acidic pH stress, however, no clear trends were observed over time.
(107) The overall lowest impact of acidic stress on the reduction of basic variants was observed for variants mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2). This result is in good agreement with the results obtained by icIEF in freeze-thaw study. In the SEC analysis, all variants were observed to accumulate some aggregates upon exposure to acidic stress which was related to a decrease of the % main peak, however, no clear trends were observed over time. In general, the accumulation of aggregates was about 9 times greater for IgG4 variants as compared to the IgG1 variants. The accumulation of aggregates for IgG1 variants was 0.3 to 1.0% for all acidic stress samples, and for IgG4 variants the accumulation was 4.3 to 7.8% for stress samples, which is significantly higher (see Table 9).
(108) In conclusion, it was determined that the highest resistance to acidic stress was observed for variants mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2).
(109) d. Heat Stress
(110) Samples for each variant were subjected to heat stress in a heat block at 50? C. for 3 days, 1 week, and 2 weeks and then compared to the set of general control samples stored at 2-8? C. Appearance testing of the samples was performed periodically and there was no observed evidence of phase separation, change in opacity, or precipitation. All samples were observed to be clear, colorless and free of visible particulates for all timepoints (data not shown). In addition, there was no significant change in protein concentration as determined by UV method (data not shown).
(111) The effects of heat stress on the panel of variants were evaluated by SEC, R-CGE, NR-CGE and icIEF methods. The results revealed that all variants were susceptible to heat stress.
(112) In the SEC analysis, clear trends for increasing aggregate concentration were observed as a function of time for all tested variants. Significantly higher accumulation of aggregates was observed for IgG4 variants compared to IgG1. After two weeks, the increase of % total aggregates ranged from 33.7 to 44.7% for the IgG4 variants but only 13.0 to 18.2% for the IgG1 variants (see Table 9). Additionally, the mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2) variants accumulated less aggregate than the control mAb3 (IgG1 20.1 VH3/VK1).
(113) In the R-CGE analysis, clear trends for decreasing purity (defined as the decrease of the % of LC+HC) were observed as a function of time for all tested variants, however, there were no significant differences observed in the stability between them. The level of samples degradation was comparable for all tested antibodies (data not shown). Similarly, NR-CGE data revealed clear trends for decreasing purity (defined as the decrease of the main peak) as a function of time for all tested variants. Significantly lower purity was observed for IgG4 variants compared to IgG1 (data not shown). After two weeks the % purity was decreased by 34.8 to 41.7% for the IgG4 variants but only 20.0 to 30.0% for the IgG1 variants (data not shown). Additionally, mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2) variants exhibited lower degradation than mAb3 IgG1 20.1 VH3/VK1. For the IgG1 antibodies, the reduction in main peak was primarily accompanied by an increase of front main peak impurities (fragments). For IgG4 antibodies, however, the accumulation of both front main peak impurities (fragments) and back main peak impurities (aggregates) were observed as a function of decreasing main peak (data not shown). This result is in good agreement with the SEC data where the significantly higher concentration of aggregates was observed for IgG4 antibodies compared to IgG1.
(114) The 1-week samples for the IgG4 variants were too degraded for analysis by icIEF, and the 2-week samples for all variants were too degraded for analysis. Therefore, only 3 days data were used to evaluate changes in charge heterogeneity between tested antibodies. The difference in purity as determined by % main peak was 12.4 to 14.4% difference for IgG1 samples and 16.0 to 16.6% for IgG4 samples (data not shown).
(115) Overall, it was demonstrated that degradation upon heat stress was greater for IgG4 variants as compared to IgG1 variants, and this was observed at all time points. The analysis indicates that mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2) variants are the least susceptible to heat stress and this result is consistent with data obtained for freeze-thaw and acidic pH stresses.
(116) Selection of the Best Candidate in Terms of Developability Properties
(117) Regarding the productivity in cell lines, the best results were obtained with the humanized variant mAb1 (7.2 VH2/VK1), with a viable cell density raising 54?10.sup.6 viable cells/ml at 10 days for mAb1.
(118) In the thermal stability study, samples were assessed in the standard matrix by DSF analysis to determine T.sub.onset, T.sub.m and T.sub.agg for each candidate. No variation in T.sub.onset and T.sub.m1 were observed, and T.sub.agg was greater than 70? C. for all variants. The results indicate that mAb1, mAb2, mAb4, and mAb5 demonstrate comparable thermal stability.
(119) In the forced degradation study, samples were exposed to agitation, freeze-thaw, acidic pH and heat stresses. The results indicate that the panel of candidate variants were susceptible to degradation at varying extents under relevant stress conditions: No significant response to agitation stress was observed by any of the analytical methods. No significant response to freeze-thaw stress was observed by SEC or CGE methods, however icIEF analysis revealed differences in charge heterogeneity between tested variants and showed that mAb1 (IgG1 7.2 VH2N/VK1) and mAb2 (IgG1 7.2 VH2N/VK2) variants exhibited the highest resistance to changes caused by freeze-thaw stress. A response to acidic pH stress was observed by icIEF and SEC, wherein all candidates accumulated some impurities upon exposure. The mAb1 (IgG1 7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2) variants exhibited the highest resistance to acidic pH stress. The most significant stress response observed was to sample storage at 50? C. SEC, NR-CGE, and icIEF analyses revealed a significant trend of decreasing sample purity over time. The observed decrease in purity was consistently greater for IgG4 variants as compared to IgG1 variants. In the group of IgG1 variants, the mAb1 (7.2 VH2/VK1) and mAb2 (IgG1 7.2 VH2/VK2) exhibited the highest resistance to acidic heat stress.
(120) The results of the developability properties of the different humanized candidates are summarized in the Table 9 below:
(121) TABLE-US-00009 TABLE 9 mAb1 humanized candidate has higher developability properties as compared to all other tested humanized variants Heat Stress- SEC analysis Freeze-thaw Acidic (Difference stress- Stress-SEC in % Agg as Pool growth Decrease of analysis measured (viable cell % basic (increase by SEC density at 10 variants.sup.1 in % analysis days/ml) and (3 cycles, aggregates after 2 productivity 6 cycles and at pH 3.5 weeks at Name Candidate (?g/ml) 10 cycles) after 24 h) 50? C.) mAb1 7.2 VH2/ 54 ? 10.sup.6 1.2 0.2 13.9 VK1 IgG1 550 ?g/ml 1.2 1.0 mAb2 7.2 VH2/ 30 ? 10.sup.6 0.9 0.8 13.1 VK2 IgG1 330 ?g/ml 0.6 1.3 mAb3 20.1 VH3/ n.d 2.1 0.3 18.3 VK1 IgG1 2.0 3.4 mAb4 7.2 VH2/ 44 ? 10.sup.6 1.9 4.4 33.6 VK1 IgG4 780 ?g/ml 1.9 3.0 mAb5 7.2 VH2/ 39 ? 10.sup.6 1.6 5.1 35.4 VK2 IgG4 475 ?g/ml 1.8 2.8 mAb6 20.1 VH3/ 38 ? 10.sup.6 2.5 6.3 44.6 VK1 IgG4 430 ?g/ml 2.4 4.7 .sup.1Change in % Basic Variants was calculated based on non-rounded results, subtracting the % basic variants of the stressed samples from that of the respective control sample.
(122) As a conclusion, mAb1 variant showed the best results in terms of developability and was selected as the lead candidate.
(123) Functional Properties of Humanized mAb 7.2 Variants as Monovalent and Bivalent Molecules
(124) 1. Preparation of Fab and Fab.sub.2 Fragments
(125) a. Pepsin Digestion for F(ab).sub.2 Generation
(126) Immobilized pepsin in 50% slurry (Thermo Scientific kit, Cat. N?44988) was buffer-exchanged into digestion buffer (20 mM Sodium acetate pH 4.4) by spinning down the slurry at 5000 g 2 min. The supernatant was discarded and slurry resuspended in 1 mL digestion buffer followed by another spin at 5000 g 2 min. This step was repeated additional four times (five resin washes in total). The resin was then resuspended in digestion buffer up to the original slurry volume.
(127) mAb 4, mAb 5 and mAb 6 (7.2 VH2/VK1, 7.2 VH2/VK2 or 20.1 VH3/VK1 IgG4 variants) were buffer-exchanged into digestion buffer using 5 or 10 mL Zeba Spin column (scale dependent) and concentrated to 3 mg/mL using Vivaspin concentrator (10,000 MWCO) according to manufacturer protocol.
(128) mAbs 4, 5 and 6 at 3 mg/mL (previously buffer exchanged in digestion buffer) were mixed with pepsin immobilized on resin and incubated at 37? C. rotating, 1.5-2 hours.
(129) Digestion mixtures were spin down at 5000 g 2 min. The supernatants were removed and filtered into a fresh tube using an appropriate syringe and 0.22 ?m small filter (Merck Millipore, Millex Cat n? SLGV004SL). The resins were washed with 1 mL PBS, spin at 5000 g 1 min. The supernatants were removed, filtered and pooled with previous supernatants. The wash step was repeated.
(130) Digested and filtered supernatants were purified using HiLoad 16/600 200 ?g size exclusion column using 10 mM Sodium acetate, 100 mM NaCl, pH 5.5 as a mobile phase. The fractions corresponding to eluted F(ab).sub.2 fragment are pooled, concentrated and sterile filtered.
(131) F(ab).sub.2 fragments were analyzed by SDS-PAGE, analytical SEC and OD.sub.280nm reading.
(132) b. Papain Digestion for Fab Generation
(133) Immobilized papain in 50% slurry (Thermo Scientific kit, Cat. N? 20341) was buffer-exchanged into digestion buffer (20 mM Sodium Phosphate, 10 mM EDTA, 150 mM Cysteine pH 7.0) by spinning down the slurry at 5000 g 2 min. The supernatant was discarded and slurry resuspended in larger volume of digestion buffer followed by another spin at 5000 g 2 min. This step was repeated additional four times (five resin washes in total). The resin was then resuspended in digestion buffer up to the original slurry volume.
(134) mAb 4, mAb 5 and mAb 6 (7.2 VH2/VK1, 7.2 VH2/VK2 or 20.1 VH3/VK1 IgG4 variants) were buffer-exchanged into digestion buffer using 5 or 10 mL Zeba Spin column (scale dependent) and concentrated to 3 mg/mL using Vivaspin concentrator (10,000 MWCO) according to manufacturer protocol.
(135) mAbs 4, 5 and 6 at 3 mg/mL (previously buffer exchanged in digestion buffer) were mixed with papain immobilized on resin and incubated at 37? C. rotating, 42 hours.
(136) Digestion mixtures were spin down at 5000 g 2 min. The supernatants were removed and filtered into a fresh tube using an appropriate syringe and 0.22 ?m small filter (Merck Millipore, Millex Cat n? SLGV004SL). The resins were washed three times with PBS, spin at 5000 g 1 min, collecting and pooling at each cycle the supernatant. Pooled fractions were then filtered into a fresh tube using an appropriate syringe and 0.22 ?m small filter. Digested and filtered supernatants were first buffer-exchanged into 1?DPBS, pH 7.4, then first purified using protein A column to remove Fc and undigested mAbs, followed by polishing step using size-exclusion (SEC) column with 10 mM sodium acetate, 100 mM NaCl, pH 5.5 as a mobile phase. The fractions corresponding to eluted Fab fragments were pooled, concentrated and sterile filtered.
(137) Fab fragments were analyzed by SDS-PAGE, analytical SEC and OD.sub.280nm reading.
(138) 2. Affinity Measurement of Humanized 7.2 and 20.1 Fab, F(ab).sub.2 Fragments and IgG Format Using Biacore
(139) In order to assess the affinity of lead humanized 7.2 and 20.1 antibodies for BTN3A1 in their different formats (Fab, F(ab).sub.2 and IgG), single cycle kinetic analysis was performed using a Biacore T200 (serial no. 1909913) instrument running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden). All single cycle kinetic experiments were run at 25? C. with HBS-P+ running buffer (pH 7.4) containing 0.1% BSA (GE Healthcare, Little Chalfont, UK).
(140) BTN3A1 His-tagged antigen (Sino Biological, Beijing, China) was diluted in running buffer to a final concentration of 0.4 ?g/ml. At the start of each cycle, BTN3A1-His was captured onto Fc2 of CM5 sensor chip pre-coupled using a His capture kit (GE Healthcare, Little Chalfont, UK) with standard amine chemistry at a flow rate of 10 ?l/min. An immobilization level (RL) of ?34 RU, 16 RU or 11 RU, the different theoretical values to obtain a RMax of ?50 RU was used for the analytes Fab, F(ab).sub.2 and IgG respectively. The surface was then allowed to stabilize. Single cycle kinetic data was obtained with the purified samples (Fab, F(ab).sub.2 and IgG) at a flow rate of 40 ?l/min to minimize any potential mass transfer effects. The signal from the reference channel Fc1 (no antigen capture) was subtracted from that of Fc2 to correct for differences in non-specific binding to the reference surface. The signal for BTN3A1-His blank runs (no analyte) were subtracted to correct for differences in surface stability. The association phase for the five injections of increasing concentrations was monitored for 240 seconds each time and a single dissociation phase was measured for 1400 seconds following the last injection of analyte. Regeneration of the chip surface was conducted using two injections of 10 mM glycine-HCL pH 1.5 followed by a stabilization period of 240 seconds. Raw sensorgrams were fitted with a 1:1 model for Fab samples and with a bivalent analyte model for F(ab).sub.2 and IgG samples in agreement with the different valences of the analytes. The kinetic constants were calculated for each variant (see Tables 10, 11 and 12).
(141) TABLE-US-00010 TABLE 10 Single cycle kinetic IgG analysis IgG (bivalent fitting) K.sub.a1 K.sub.a2 Sample (1/Ms) (1/Ms) K.sub.d 1 (1/s) K.sub.d 2 (1/s) R.sub.max (RU) Chi.sup.2 (RU.sup.2) mAb 1 (7.2 5.30 ? 10.sup.4 4.21 4.63 ? 10.sup.?4 4.40 ? 10.sup.1 69.6 0.063 VH2/VK1) mAb 2 (7.2 5.97 ? 10.sup.4 2.01 ? 10.sup.?3 4.03 ? 10.sup.?4 2.41 ? 10.sup.?2 68.0 0.144 VH2/VK2) mAb 3 (20.1 5.63 ? 10.sup.4 5.47 ? 10.sup.?5 3.79 ? 10.sup.?3 8.60 ? 10.sup.?5 56.1 0.042 VH3/VK1)
(142) TABLE-US-00011 Single cycle kinetic F(ab).sub.2 analysis F(ab).sub.2 (bivalent fitting) K.sub.a1 K.sub.a2 Sample (1/Ms) (1/Ms) K.sub.d 1 (1/s) K.sub.d 2 (1/s) R.sub.max (RU) Chi.sup.2 (RU.sup.2) 7.2 VH2/VK1 8.33 ? 10.sup.4 6.10 6.66 ? 10.sup.?4 3.24 ? 10.sup.1 78.2 0.033 F(ab).sub.2 7.2 VH2/VK2 1.46 ? 10.sup.5 3.40 3.94 ? 10.sup.?4 5.46 ? 10.sup.1 58.9 0.126 F(ab).sub.2 20.1 VH3/VK1 9.23 ? 10.sup.4 5.03 ? 10.sup.?5 4.16 ? 10.sup.?3 .sup.8.10 ? 10.sup.?5 61.5 0.326 F(ab).sub.2
(143) TABLE-US-00012 TABLE 12 Single cycle kinetic Fab analysis Fab (1:1 fitting) Sample K.sub.a (1/MS) K.sub.d (1/S) K.sub.D (nM) R.sub.max (RU) Chi.sup.2 (RU.sup.2) 7.2 1.98 ? 10.sup.5 6.39 ? 10.sup.?4 3.22 66 0.114 VH2/VK1 Fab 7.2 2.22 ? 10.sup.5 6.69 ? 10.sup.?4 3.01 61 0.091 VH2/VK2 Fab 20.1 1.76 ? 10.sup.6 3.57 ? 10.sup.?2 20.30 55 1.330 VH3/VK1 Fab
3. Selection of the Best Candidate in Terms of Affinity Properties
(144) 20.1 and 7.2 humanized variants showed significant differences in terms of affinity properties. These differences can be observed with IgG and F(ab).sub.2 formats, but the gap is even higher with Fab fragments: indeed, 20.1 showed a mean K.sub.D of 20.30 nM while 7.2 showed a mean K.sub.D of 3.22 (VH2/VK1) or 3.01 (VH2/VK2).
(145) 4. mAb 1 Binding Avidity to Primary T Cells and Other Cell Lines
(146) Next, mAb1 binding avidity has been evaluated by flow cytometry on human primary T cells as well as on various cell lines including WT and BTN3A knock-out reconstituted with BTN3A1, BTN3A2 or BTN3A3 isoforms individually (Data not shown). EC.sub.50 values obtained for each tested cell type are summarized in Table 13.
(147) Data obtained in BTN3A KO cells clearly indicated that mAb1 binds specifically to its target. In addition, reconstitution with individual isoforms confirmed that mAb1 recognizes BTN3A1, BTN3A2, and BTN3A3 with comparable avidity. All other tested cells appeared positive for mAb1 binding with a range of EC50 values going from 9.6 nM for Burkitt's lymphoma Daudi cell line to 112.3 nM for colorectal adenocarcinoma cell line HT29.
(148) TABLE-US-00013 TABLE 13 mAb1 Binding Avidity on Human Cells. EC.sub.50 EC.sub.50 ?g/mL (?10.sup.?9M) Cells Description Gating (+/?sem) (+/?sem) Human HV Primary (n = 6) CD3+ 5.25 34.9 PBMC (+/?0.57) (+/?3.8) Daudi Burkitt's lymphoma NA 1.44 9.6 (n = 5) (+/?0.16) (+/?1.04) L-IPC Pancreatic Ductal NA 26.26 175.1 (PDAC087T) Adenocarcinoma (n = 4) (+/?4.4) (+/?29.3) HT29 Colorectal NA 16.84 112.3 adenocarcinoma (n = 3) (+/?3.1) (+/?20.6) A549 Lung carcinoma (n = 6) NA 6.53 43.56 (+/?1.8) (+/?12.3) HUVEC Endothelial (n = 2) NA 12.8 85.2 (+/?1.98) (+/?13.2) HEK293T WT Embryonic kidney NA 6.2 41.3 (n = 3) (+/?0.44) (+/?2.95) HEK293T Embryonic kidney; Full NA NA NA BTN3KO Knock-out for BTN3A1, A2, A3 (n = 3) HEK293T Embryonic kidney; Full CFP+ 2.4 15.9 BTN3KO + Knock-out for BTN3A1, (+/?0.34) (+/?2.3) 3A1-CFP A2, A3 transiently transfected with BTN3A1-CFP (n = 4) HEK293T Embryonic kidney; Full CFP+ 1.88 12.5 BTN3KO + Knock-out for BTN3A1, (+/?0.39) (+/?2.6) 3A2-CFP A2, A3 transiently transfected with BTN3A2-CFP (n = 5) HEK293T Embryonic kidney; Full CFP+ 1.75 11.68 BTN3KO + Knock-out for BTN3A1, (+/?0.11) (+/?0.72) 3A3-CFP A2, A3 transiently transfected with BTN3A3-CFP (n = 5) NA: Not applicable
5. mAb1 has No Off-Target Binding
(149) The potential for off-target binding of mAb1 on other non-BTN3A molecules was assessed by the Retrogenix technology. This work aimed to demonstrate the absence of off-target binding by screening a cell array expressing >5000 human membrane receptors or secreted proteins expressed at the surface of HEK293 cells (Retrogenix platform).
(150) Investigation of the level of binding of mAb1 to untransfected HEK293 cells, and to cells over-expressing BTN3A1, before or after cell fixation, showed 5 ?g/ml on fixed cells to be a suitable screening condition. Under this condition, mAb1 was screened for binding against human HEK293 cells, individually expressing 5528 human proteins, comprising of cell surface membrane proteins, and cell surface-tethered secreted proteins. This revealed ten primary hits.
(151) Each primary hit was re-expressed, along with two control receptors (CD20 and EGFR), and re-tested with 5 ?g/ml of mAb1, 5 ?g/ml of an isotype control antibody, and other positive and negative control treatments. After removing five non-specific hits, there remained five specific interactions for the test antibody. All 5 specific hits were BTN3A related; two isoforms of BTN3A1, two isoforms of BTN3A2 and one isoform of BTN3A3.
(152) The study conclusions are that, no off-target interactions for mAb1 were identified, indicating high specificity of mAb1 for its BTN3A epitope.
(153) 6. mAb1 Mediates V?9V?2 T Cell Activation and Tumour Cell Killing
(154) Originally, mouse anti-BTN3A mAbs were shown to trigger BTN3A recognition by V?9V?2 T cells, mediating activation leading to (i) proliferation of this specific subset in human PBMCs, (ii) production of cytokines (IFN? and TNF?), and (iii) the cytolysis of infected or transformed target cells (e.g., By Perforin, granzymes, TRAIL) (Harly et al, Blood, 2012 & Benyamine, A. et al. 2016, Oncoimmunology 5, e1146843).
(155) Without being bound by any particular theory, a proposed mechanism of action of mAb1 is that its binding to BTN3A expressed at the surface of a tumour target cell triggers a conformational change that allows its signalling to its counter-receptor on V?9V?2 T cells. The activity of anti-BTN3A antibodies is routinely assessed using an in vitro assay based on co-culture of a tumour cell line (the target) with primary human V?9V?2 T cells (the effector) previously expanded from PBMCs of healthy donors for 10 to 14 days, in presence of rHuIL-2 (200 UI/mL) and aminobisphosphonates (Zometa, 1 ?M). At the end of the expansion phase, the purity of V?9V?2 T cells is assessed by flow cytometry, and these cells are then frozen for future use. The day before the experiment, expanded V?9V?2 T cells are thawed and cultured overnight with 200 UI/mL rHuIL-2 to maintain in vitro survival. After co-culture, V?9V?2 T cell activation is monitored either by flow cytometry detection of CD107a/b expression on ?? T cells, or by quantifying Caspase3/7 activation, as a measure of target cell killing.
(156) First, human V?9V?2 T cells expanded from 3 different healthy donors PBMCs were co-cultured with the Daudi cell line (ATCC-CCL213; Burkitt's lymphoma) with increasing concentrations of mAb1. After 4 hours, cells were analysed for V?9V?2 T cell expression of CD107a/b by flow cytometry. Results showed a concentration related increase in the percentage of V?9V?2 T cells expressing CD107a/b, with a mean EC.sub.50 of 0.89 nM (+/?0.39) (
(157) 7. mAb1 Enhances V?9V?2 T-Cell Killing of a Wide Array of BTN3A Expressing Human Cell Lines, Irrespective of their Tissue Origin
(158) a. Material & Methods.
(159) Tumoral Cell Culture
(160) HL-60 are human promyeloblast cell line derived from acute promyelocytic leukemia. Daudi is a human B lymphoblastic cell line derived from Burkitt' lymphoma. Jurkat is an acute T cells leukemia cell line.
(161) HT-29 and HCT116 are human epithelial cells derived from colorectal adenocarcinoma.
(162) PC3 and DU145 were derived from prostate carcinoma (metastatic site, bone and brain respectively).
(163) SUM159 and MDA-MB-231 are triple negative breast cancer (TNBC) cell lines.
(164) HL60, Daudi, Jurkat, DU145 and MDA-MB-231 cells were cultured in RPMI Glutamax, 10% FBS; 1 mM Sodium Pyruvate at 37? C./5% CO2. PC3 and HT-29 were cultured in DMEM 10% FBS, 1 mM Sodium Pyruvate at 37? C./5% CO2. HCT116 were cultured in McCoy 5a medium 10% SVF. SUM159 were cultured in medium F12 Nut Mix 1?+ Glutamax, 5% SVF, Hydrocortisone 2 mg/ml, Insulin humalog 2 mg/ml and Non-Essential-Amino-Acids.
(165) In Vitro V?9V?2 T-Cells Expansion.
(166) Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation of peripheral blood obtained from the Etablissement Fran?ais du Sang Provence Alpes C?te d'Azur (France). To expand V?9V?2 T cells, 50.106 PBMCs were resuspended at 1.5?106 cells/ml in RPM11640 supplemented with 10% FBS and 1% sodium pyruvate in 75 cm2 flasks for 10 to 14 days, in presence of rHuIL-2 (200 UI/mL) and aminobisphosphonates (Zoledronate, 1 ?M). From day 5, rHuIL-2 was renewed every 2 or 3 days and cells were kept at 1?106/ml. At the end of the expansion phase, the purity of V?9V?2 T cells was assessed by flow cytometry, and if the number of V?9V?2 T cells reached 80% of live cells, these cells were then frozen in FBS 20% DMSO for future use.
(167) V?9V?2 T-Cell Purification from Human PBMC.
(168) Fresh human PBMC were resuspend at 50.106 cells/ml in PBS+2% FBS+1 mM EDTA. Vg9Vd2 were isolated using EasySep? Human Gamma/Delta T Cell Isolation Kit (Stemcell #19255) according to manufacturer instructions. At the end of the procedure, cell purity was measured by flow cytometry. Live CD3+ V?9+ cells were more than 80%.
(169) V?9V?2 T-Cells Killing Assay.
(170) 10 000 expanded or fresh V?9V?2 T-cells were co-cultured with tumoral cell lines in 96 well plates at indicated ratio (E:T 1:1 or 1:5) in RPMI 1640+glutamax, 10% FBS+1 mM NaPy in presence of mAb1 or relevant isotype control. When added to the co-culture rHuIL-2 was used at 20 IU/ml. After indicated time, ATP was measured using the Glo reagent (Promega #G7572) that generates a luminescent signal proportional to the number of live cells.
(171) b. Results
(172) In addition to a method based on caspase 3/7 staining of target cells to monitor killing upon co-culture with V?9V?2 T-cells, we developed a complementary approach based on assessment of the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells.
(173) With this assay, we monitored survival of the acute myeloid leukemia cell line HL60-WT, or -BTN3A-KO, 24 hrs after co-culture with expanded V?9V?2 T-cells (ratio E:T 1:1) in the presence of mAb1 or relevant isotype control +/?rHuIL-2 (20 IU/ml) (
(174) Next, we used this assay to monitor mAb 1-mediated V?9V?2 T-cells killing activity against BTN3A expressing cell lines from different tissues origin (Colon, breast, prostate, T lymphoma and Burkitt's lymphoma). HT29, PC3, DU145, MDA-MB-231, HCT116, SUM159, Jurkat and Daudi cells were co-cultured over-night with in-vitro expanded V?9V?2 T-cells in presence of increasing concentration of mAb1 and cell viability was measured. Results confirmed that mAb1 enhanced V?9V?2 T-cell killing of a wide array of BTN3A expressing human cell lines, irrespective of their tissue origin.
(175) TABLE-US-00014 TABLE 15 10,000 Tumoral cells were co-cultured 24 hrs with in-vitro expanded V?9V?2 T-cells (ratio E:T 1:1) in presence of different concentration of mAb1. Cell viability was measured using bioluminescent assay detecting ATP levels. Bioluminescence values ?10.sup.5 are indicated. Tumoral Tissues No mAb mAb1 0,1 ug/ml mAb1 1 ug/ml mAb1 10 ug/ml cell line origin Donor A Donor B Donor A Donor B Donor A Donor B Donor A Donor B HT29 Colon 68.0 55.1 67.6 58.1 54.2 49.2 41.0 40.0 PC3 Prostate 86.2 73.5 83.3 68.5 48.6 49.7 36.8 38.0 DU145 Prostate 75.7 69.6 65.1 64.4 33.6 41.8 22.1 26.8 MDA-MB- Breast 66.5 59.8 68.2 59.8 57.6 54.7 43.4 44.4 231 HCT116 Colon 114.1 102.7 102.9 96.3 63.6 68.3 53.4 59.4 SUM159 Breast 84.7 90.8 54.1 58.5 20.7 25.2 16.4 19.2 Jurkat T 26.8 26.4 3.7 4.1 2.6 9.7 4.1 1.6 lymphoma Daudi Burkitt's 17.3 9.7 6.2 5.0 7.2 5.3 6.0 6.1 lymphoma
8. mAb1 Improves V?9V?2 T Cell Therapy in a Mouse Model Engrafted with Human AML Cell Lines.
(176) As mAb1 target BTN3A is not expressed in rodents, and the Vg?V?2 T cell sub-population is specific to primates, experimental proof of concept of V?9V?2 T cell anti-tumoral activity is usually tested in immunocompromised NSG mice engrafted with human tumour cell lines which express BTN3A, and adoptively transferred with V?9V?2 T cells from health human donors (the V?9V?2 T cells in such reconstitutions are allogenic to the tumour cell line). These models have been widely used to validate the anti-tumoral therapeutic potential of V?9V?2 T cells in a broad range of solid, as well as haematological, malignancies (Pauza, C. D. et al. 2018, Immunol. 9).
(177) In addition, the murine anti-human BTN3A antibody, m20.1, administered to mice which had received human V?9V?2 T cells, was described to enhance animal survival, and decrease the leukemic burden, in blood and bone marrow of AML bearing NSG mice (benyamine et al 2016, see above). In this model, m20.1 was injected in combination with RLI, an rHuIL-15/IL-15Ra fusion protein that was shown to expand antitumor lymphocyte subsets, and to improve their survival in mice, leading to better anti-tumour activity of the transferred human V?9V?2 T cells.
(178) For further characterisation of the efficacy of mAb1 in in vivo mouse models, we investigated the potential of human V?9V?2 T cells transfer combined with mAb1 to delay tumour growth and improve animal survival in AML bearing NSG mice.
(179) a. U937 Model.
(180) Healthy 6-8-weeks-old female mice (n=30) received 0.2?10.sup.6 luciferase transduced-U937 cells on Day 0, as described in Gertner-Dardenne, J. et al. 2012. J. Immunol. 188, 4701-4708. At day 1, tumoral load was evaluated using bioluminescent imaging and mice were randomly assigned in six groups to receive intravenous injections of in-vitro expended human V?9V?2 T cells on Day 1 (3?10.sup.6 cells) alone or combined to anti-BTN3A mAb1 or relevant isotype control (10 mg/kg, 200 ug/mice). Treatment was repeated at day 7. In group 2 and 4 antibodies were also administrated at day 4 and 10. As complexes of rHuIL-15/rHuIL15-Ra allows the proliferation of V?9V?2 T cells (J. Immunol. (2006) 177, 6072-608), these complexes were administered together with V?9V?2T cells.
(181) All groups are described in Table 16.
(182) The hIL-15/IL-15R-Fc complexes were pre-complexed at room temperature (RT) for 30 minutes before injection (0.2 ug hIL-15+1.2 ug IL-15R-Fc per mice) and mixed with V?9V?2 T cells prior injection. The final volume for each injection was 100 ?l. Treatment mAbs were injected 4 hours prior to V?9V?2 T cell engraftment.
(183) Our results confirmed that V?9V?2 T cells plus-rHuIL-15/rHuIL15-R? infusion decrease the tumour burden. This effect was even more striking and was associated with a significant increase of survival when anti-BTN3A mAb1 was added along with V?9V?2 T cells (Table 16 and 17
(184) Table). Importantly, Cytarabine (Ara-C), one of the most effective drugs for the treatment of acute myeloid leukemia, used in this very aggressive mouse model (10 mg/kg in NSG mice bearing U937 tumour) improved mice survival for 2 to 3 days (?10%), as observed with mAb1 combine to human V?9V?2 T cell transfer.
(185) These data highlight the potent anti-leukemic effect exerted by anti-BTN3A mAb combined to V?9V?2 T cells immunotherapy in vivo.
(186) TABLE-US-00015 TABLE 16 U937 AML mice model: group description. Group Day 1 Day 4 Day 7 Day 10 1 No No No No 2 V?9V?2 T cells hIgG1 V?9V?2 T cells hIgG1 (3 ? 106 cells) + (10 mg/kg) (3 ? 106 cells) + (10 mg/kg) IL-15a + hIgG1 IL-15a + hIgG1 (10 mg/kg) (10 mg/kg) 3 V?9V?2 T cells No V?9V?2 T cells No (3 ? 106 cells) + (3 ? 106 cells) + IL-15a + mAb1 IL-15a + mAb1 (10 mg/kg) (10 mg/kg) 4 V?9V?2 T cells mAb1 V?9V?2 T cells mAb1 (3 ? 106 cells) + (10 mg/kg) (3 ? 106 cells) + (10 mg/kg) IL-15a + mAb1 IL-15a + mAb1 (10 mg/kg) (10 mg/kg) 5 V?9V?2 T cells No V?9V?2 T cells No (3 ? 106 cells) + (3 ? 106 cells) + IL-15a + hIgG1 IL-15a + hIgG1 (10 mg/kg) (10 mg/kg) 6 V?9V?2 T cells No V?9V?2 T cells No (3 ? 106 cells) + (3 ? 106 cells) + IL-15a + m20.1 IL-15a + m20.1 (10 mg/kg) (10 mg/kg) a: rHu-IL-15/rHu-IL-15R? complexes.
(187) TABLE-US-00016 TABLE 17 Anti-BTN3A (mAb1) Activating mAb has Anti-leukaemic Activity In Vivo in AML Xenograft Models. Bioluminescence Data of U937 model. Mouse Day 1 Day 7 Day 14 Day 19 Group 1 1 1.10E+03 8.33E+04 1.06E+07 Dead PBS 2 7.04E+03 2.50E+05 6.14E+07 Dead 3 7.57E+03 8.40E+04 2.41E+07 Dead 4 9.23E+03 2.55E+05 6.48E+07 Dead 5 1.37E+04 1.36E+05 3.58E+07 Dead Mean 7.73E+03 1.62E+05 3.93E+07 NA Group 2 1 3.24E+03 1.48E+04 5.23E+06 1.82E+08 LT?? + hIgG1 2 5.61E+03 1.39E+05 3.23E+07 Dead (Day 1, 4, 7, 10) 3 8.76E+03 3.44E+04 1.60E+07 2.23E+08 4 9.87E+03 2.61E+04 1.66E+07 Dead 5 1.03E+04 2.90E+05 3.14E+07 3.19E+08 Mean 7.56E+03 1.01E+05 2.03E+07 NA Group 3 1 3.38E+03 5.38E+03 1.89E+06 1.87E+08 LT?? + mAb1 2 5.22E+03 3.68E+04 3.95E+06 2.04E+08 (Day 1, 7) 3 7.49E+03 8.44E+04 1.23E+07 1.05E+08 4 7.73E+03 6.67E+04 4.93E+06 1.79E+08 5 1.29E+04 1.55E+04 5.32E+06 8.47E+07 Mean 7.34E+03 4.18E+04 5.68E+06 1.52E+08 Group 4 1 4.08E+03 1.32E+03 2.14E+06 8.98E+07 LT?? + mAb1 2 5.17E+03 7.58E+04 1.55E+06 1.79E+08 (Day 1, 4, 7, 10) 3 9.21E+03 2.82E+04 6.82E+06 1.24E+08 4 8.97E+03 4.00E+04 6.38E+06 1.91E+08 5 1.00E+04 4.02E+04 3.65E+06 9.26E+07 Mean 7.49E+03 3.71E+04 4.11E+06 1.35E+08 Group 5 1 4.15E+03 4.01E+04 2.96E+06 1.19E+08 LT?? + mIgG1 2 4.53E+03 7.96E+04 4.91E+06 2.40E+08 (Day 1, 7) 3 9.72E+03 1.66E+05 1.46E+07 2.97E+08 4 8.92E+03 6.36E+04 1.35E+07 1.47E+08 5 1.02E+04 3.97E+04 1.39E+07 1.91E+08 Mean 7.50E+03 7.78E+04 9.97E+06 1.99E+08 Group 6 1 4.34E+03 8.51E+04 3.55E+06 1.57E+08 LT?? + m20.1 2 4.42E+03 3.05E+04 4.28E+06 1.87E+08 (Day 1, 7) 3 8.66E+03 6.62E+04 4.24E+06 5.55E+07 4 8.90E+03 3.05E+04 2.49E+06 7.32E+07 5 1.17E+04 1.62E+04 1.73E+06 9.61E+07 Mean 7.60E+03 4.57E+04 3.26E+06 1.14E+08
(188) TABLE-US-00017 TABLE 18 Anti-BTN3A (mAb1) Activating mAb has Anti-leukaemic Activity In Vivo in AML Xenograft Models. Survival after U937 cell line engraftment. Day of Day of Mouse death Mouse death Group 1 1 18 Group 4 1 20 PBS 2 18 LT?? + mAb1 2 22 3 19 (Day 1, 4, 7, 10) 3 23 4 19 4 23 5 19 5 24 Median 19 Median 23 Group 2 1 20 Group 5 1 20 LT?? + hIgG1 2 20 LT?? + mIgG1 2 20 (Day 1, 4, 7, 10) 3 21 (Day 1, 7) 3 21 4 21 4 21 5 22 5 22 Median 21 Median 21 Group 3 1 21 Group 6 1 23 LT?? + mAb1 2 21 LT?? + m20.1 2 24 (Day 1, 7) 3 22 (Day 1, 7) 3 24 4 24 4 24 5 24 5 24 Median 22 Median 24
(189) b. MOLM14 Model.
(190) The in vivo efficacy of mAb1 against MOLM14, an AraC resistant human AML derived cell line, was evaluated in a xenograft model using NSG mice transplanted with the human tumor cell line, and human V?9V?2 T cells. The goal of this study was to confirm the effect of repeated iv injections of human V?9V?2 T cells in combination with mAb1 on tumor growth and on mice survival.
(191) Six to eight-week-old female NSG mice were injected intravenously (iv) via the tail vein at day 0 with 0.2?106 per mouse in a volume of 100 ?l of MOLM14 (CVCL_7916) cells expressing luciferase (luc2). Bioluminescence analysis was performed at day 0 using PhotonIMAGER (Biospace Lab) following addition of endotoxin-free luciferin (30 mg/kg) and mice were randomized in homogeneous groups of 7 mice based on the strength of the bioluminescence signal. 3?106 human in-vitro expanded V?9V?2 T cells and hIL-15/IL-15R complexes were iv injected at days 1, 8, 15, 22 and 29. mAb1 or hIgG1 were iv injected at days 1, 5, 8, 12, 15, 19, 22, 26 and 29.
(192) The different experimental groups are summarized in Table 19.
(193) The hIL-15/IL-15R-Fc complexes were pre-complexed at room temperature (RT) for 30 minutes before injection (0.2 ug hIL-15+1.2 ug IL-15R-Fc per mice) and mixed with V?9V?2 T cells prior injection. The final volume for each injection was 100 ?l. Treatment mAbs were injected 4 hours prior to V?9V?2 T cell engraftment.
(194) Bioluminescence signal emitted by the MOLM14 cells was measured at days 0, 7, 14, 21 and 28 after cell injection to follow tumor growth. Blood sampling was conducted at day 19 to assess the number of circulating MOLM14 cells by flow cytometry. Red cell lysis was performed before staining. mCD45+ murine cells were excluded from the analysis, MOLM14 tumor cells were detected by their GFP expression. Acquisition was performed on a LSRII SORP cytometer (Becton Dickinson) and analysis was performed using the FlowJo software. Daily monitoring of mice for symptoms of disease (significant weight loss, ruffled coat, hunched back, weakness, and reduced mobility) determined the time of killing for injected animals with signs of distress.
(195) As shown in Table 20, V?9V?2 T cells injected with irrelevant control isotypes (hIgG1) do not significantly control tumor growth. In contrast, as shown by lower bioluminescence signals, tumor growth was strongly reduced when anti-BTN3A mAb1 was administered sequentially with human V?9V?2 T cells. Results also showed a significant decrease in the number of circulating blasts (assessed by flow cytometry in peripheral blood) at day 19 of the protocol (Table 20). Importantly, the anti-BTN3A mAb-dependent decrease in tumor growth leads a significant improvement of 45% in mice survival (as compared to the respective isotype control) (Table 22).
(196) TABLE-US-00018 TABLE 19 MOLM14 mice model: group description Group Number of animals Treatment at day 7 and 14 1 6 PBS 2 7 V?9V?2 T cells (3 ? 106 cells) + IL15/IL15R-Fc + hIgG1 (10 mg/kg) 3 7 V?9V?2 T cells (3 ? 106 cells) + IL15/IL15R-Fc + mAb1 (10 mg/kg)
(197) TABLE-US-00019 TABLE 20 MOLM14 mice model: bioluminescence measurement at day 0, 7, 14, 21 and 28 after tumor cell engraftment. Mouse Day 0 Day 7 Day 14 Day 21 Day 28 Group 1 1 5.1E+03 3.1E+04 1.1E+06 8.0E+06 Dead PBS 2 1.4E+04 1.1E+04 2.5E+05 2.2E+06 Dead 3 8.0E+03 3.1E+04 1.6E+06 8.8E+06 Dead 4 7.2E+03 2.5E+04 7.4E+05 2.6E+06 Dead 5 7.7E+03 1.6E+04 4.5E+05 1.6E+06 Dead 6 4.0E+03 2.5E+04 1.6E+06 1.5E+07 2.4E+07 Mean 7.59E+03 2.32E+04 9.57E+05 6.36E+06 NA Group 2 1 9.5E+03 2.0E+04 7.4E+05 4.0E+06 1.0E+07 LT??+ 2 1.3E+04 5.6E+03 5.9E+05 3.1E+06 Dead hIgG1 3 7.7E+03 1.4E+04 6.0E+05 2.7E+06 Dead 4 3.1E+03 4.5E+03 1.6E+05 5.6E+06 7.4E+06 5 1.0E+04 1.2E+04 4.7E+05 5.5E+06 1.3E+07 6 6.4E+03 3.1E+04 7.2E+05 2.8E+06 Dead 7 8.8E+03 3.8E+03 2.1E+05 1.3E+06 5.4E+06 Mean 8.45E+03 1.29E+04 4.97E+05 3.59E+06 NA Group 2 1 1.2E+04 3.0E+03 1.1E+04 8.6E+04 1.4E+06 LT??+ 2 3.9E+03 2.0E+03 2.0E+04 2.3E+04 2.0E+05 mAb1 3 1.1E+04 3.4E+03 1.2E+05 2.6E+05 2.0E+06 4 1.2E+04 2.6E+03 7.0E+03 1.7E+04 1.5E+05 5 5.7E+03 1.9E+03 1.4E+04 1.0E+05 5.2E+05 6 4.6E+03 1.3E+03 1.8E+04 2.6E+05 3.5E+06 7 5.1E+03 1.3E+03 1.2E+04 1.2E+05 2.2E+05 Mean 7.76E+03 2.19E+03 2.88E+04 1.24E+05 1.13E+06
(198) TABLE-US-00020 TABLE 21 MOLM14 mice model: Number of circulating blasts at day 19 of the protocol. Data represent number of cells/ul of blood. Mouse Day 19 Group 1 1 6.1 PBS 2 3 3 4.2 4 7.1 5 1.7 6 13.1 Group 2 1 1.2 LT?? + 2 1.1 hIgG1 3 3.3 4 1.3 5 4 6 0.9 7 0.5 Group 2 1 0 LT?? + 2 0 mAb1 3 0.02 4 0 5 0 6 0.02 7 0
(199) TABLE-US-00021 TABLE 22 MOLM14 mice model: animal survival. Day of death for each animal is indicated together with the median survival for each group. Day of Mouse death Group 1 1 23 PBS 2 26 3 27 4 27 5 28 6 31 Median 27 Group 2 1 27 LT?? + 2 27 hIgG1 3 27 4 29 5 30 6 30 7 31 Median 29 Group 3 1 39 LT?? + 2 40 mAb1 3 41 4 42 5 43 6 43 7 46 Median 42
9. mAb1 Improves V?9V?2 T Cell Therapy in a Mouse Solid Tumor Model Engrafted with Human Ovarian Cancer Cell Line SKOV-3.
(200) a. Material & Methods.
(201) In Vitro V?9V?2 T-Cells Expansion.
(202) Allogeneic human V?9V?2 T lymphocytes were amplified from peripheral blood mononuclear cells (PBMC) obtained from healthy donor blood samples provide by the Etablissment Fran?ais du Sang (EFS, Nantes, France) and after Ficoll density centrifugation (Eurobio, Les Ulis, France). First, for specific expansions of peripheral allogeneic human V?9V?2 T lymphocytes, PBMC were incubated with 3 ?M of bromohydrin pyrophosphate (BrHPP), kindly provided by Innate Pharma (Marseille, France) in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/mL streptomycin, 100 IU/mL penicillin (all from Gibco), and 100 IU/mL recombinant human IL-2 (PROLEUKIN, Novartis, Bale, Suisse). After 4 days, cultures were supplemented with 300 IU/mL IL-2. At day 21, purity was measured by flow cytometry (purity>90%). Pure human V?9V?2 T lymphocytes were further expanded using feeder cells (mixed and 35 Gy irradiated Epstein Barr Virus transformed B lymphocytes and PBMC) and PHA-L in RPMI medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 10 mg/mL streptomycin, 100 IU/mL penicillin (all from Gibco), and 300 IU/mL recombinant human IL-2 (Novartis). After three weeks, resting ex vivo expanded-V?9V?2 T lymphocytes were used for in vivo experiments.
(203) Mouse Model
(204) At day 0, 6-8 weeks of age NSG mice were injected intraperitoneally (ip) with 1?106 SKOV-3 cells (Ovarian cancer cell line, SKOV-3-luc-D3, Perkin Elmer, Waltham, MA) expressing luciferase per mouse in a volume of 100 ?L of sterile PBS. After 7 days, mice were randomized in homogeneous groups of 5 to 6 mice. At days 7 and 14, mAbs treatments, 200 ?g per mouse of mAb1 or relevant isotype control (hIgG1), were injected ip in 100 ?L of sterile PBS. 4 hours later, 5?106 of human in-vitro expanded V?9V?2 T cells were also injected ip in 100 ?L of sterile PBS per mouse.
(205) Bioluminescence signal emitted by the SKOV-3 cells was measured at days 16, 23 and 30 after tumor cell implantation to follow tumor growth. Bioluminescent imaging was realized 8 minutes after ip injection of 1.5 mg of D-luciferin (Interchim, San Diego, CA), on mice anesthetized with isoflurane 2%, with Biospace Imager (Biospace Lab, Nesles-la-Vall?e, France). Experimental endpoint was reached when mice lost 10% of their initial weight.
(206) b. Results
(207) The in vivo efficacy of mAb1 against ovarian cancer was evaluated in a xenograft model using NSG mice transplanted with the human ovarian cancer cell line SKOV-3, and human V?9V?2 T cells. The goal of this study was to assess the effect of two ip injections of human V?9V?2 T cells in combination with mAb1 on tumor growth and on mice survival.
(208) NSG mice were injected intraperitoneally (ip) at day 0 with SKOV3. After 7 days, mice were randomized in homogeneous groups according to treatments. Groups are described in Table 23:
(209) TABLE-US-00022 TABLE 23 Ovarian cancer mice model: group description Group Number of animals Treatment at day 7 and 14 1 5 PBS 2 6 V?9V?2 T cells (5 ? 106 cells) + hIgG1 (10 mg/kg) 3 6 V?9V?2 T cells (5 ? 106 cells) + mAb1 (10 mg/kg)
(210) Result showed that human V?9V?2 T cells transferred together with mAb1 significantly delayed tumor growth (Table 24) leading to a significant improvement of animal survival (Table 25). Of note, this effect was observed in the absence of pro-V?9V?2 T survival cytokines such as IL-2 or IL-15.
(211) TABLE-US-00023 TABLE 24 Ovarian cancer mice model: bioluminescence measurement 16, 23 and 30 days after tumor cell engraftment. Mouse Day 16 Day 23 Day 30 Group 1 1 87220 Dead Dead PBS 2 48486 92643 Dead 3 89032 89930 Dead 4 74242 121010 Dead 5 82132 128390 Dead Mean 7.62E+04 1.08E+05 NA Group 2 1 32060 70953 Dead LT?? + 2 82703 166009 Dead hIgG1 3 23049 51909 150537 4 85130 147390 Dead 5 18053 45097 Dead 6 41732 76461 Dead Mean 4.71E+04 9.30E+04 NA Group 2 1 43000 42420 80574 LT?? + 2 20706 43670 63418 mAb1 3 6891 47150 70070 4 11845 48139 36820 5 22958 29131 74111 6 12725 19767 Dead Mean 1.97E+04 3.84E+04 6.50E+04
(212) TABLE-US-00024 TABLE 25 Ovarian cancer mice model: animal survival. Day of death for each animal is indicated together with the median survival for each group. Mouse Day of death Group 1 1 20 PBS 2 23 3 23 4 23 5 28 Median 23 Group 2 1 23 LT?? + 2 23 hIgG1 3 28 4 28 5 28 6 35 Median 28 Group 3 1 28 LT?? + 2 35 mAb1 3 35 4 35 5 35 6 39 Median 35
10. In-Vivo Effect on Cynomolgus Monkey V?9V?2 T-Cells
(213) Because of the absence of BTN3A and the V?9V?2 T subset in rodents, and based on previous data documenting in vitro and in vivo PAg-mediated V?9V?2 T cell activation in cynomolgus macaques, the cynomolgus monkey (Macaca fascicularis) was selected as the only relevant species for nonclinical safety evaluation of mAb1.
(214) a. Material & Methods
(215) Biacore
(216) mAb1 has been assessed for binding to recombinant human or cynomolgus BTN3A1, BTN3A2 and BTN3A3 proteins (SEQ ID NO 21, 22 and 23 respectively) via Biacore multi-cycle kinetics analysis using a Biacore T200 (serial no. 1909913) instrument. mAb1 was diluted to a concentration of 2 ?g/ml in 2% BSA/PBS. At the start of each cycle, antibody was captured on the Protein A surface at a density (RL) of ?150 RU (the theoretical value to obtain an RMax of ?50 RU). Following capture, the surface was allowed to stabilize before injection of the BTN3A antigen. BTN3A was titrated in 0.1% BSA/HBS-P+ (running buffer) in a two-fold dilution range from 25 to 0.78 nM. The association phase was monitored for 420 seconds and the dissociation phase for 2000 seconds. Kinetic data was obtained using a flow rate of 50 ?l/min to minimize any potential mass transfer effects. Obtained data was fitted using a 1:1 binding model.
(217) ELISA
(218) The apparent affinity of mAb1 for Human and cynomolgus BTN3A1 was tested by ELISA. Briefly, the binding of mAb1 on the target was evaluated using recombinant BTN3A1 immobilized on a plate at 1 ?g/ml in Phosphate buffer (1?PBS), followed by a saturation step with block buffer (2% milk/PBS)). mAb1 was titrated in block buffer in a four-fold dilution range from 0.00122 to 20 ?g/ml. A secondary antibody (Goat Anti-Human Ig? chain, HRP conjugated antibody, Millipore AP502P, diluted 1:4000 in block buffer) and TMB solution were used for detection. The apparent affinity was expressed as the EC50% (the antibody concentration required to obtain 50% of the signal plateau).
(219) Binding Avidity of mAb1 to Human and Cynomolgus CD3+ Cells.
(220) After red blood cells lysis, human or cynomolgus PBL were incubated with increasing concentration of mAb1 or isotype control for 30 min at 4? C., washed two times and stained with a goat anti-human-IgG-PE conjugated secondary antibody (eBioscience? #12-4998-82). After 2 washes, cells were stained with an anti-CD3-PC3 mAb (BD Bioscience #557749) and live/Dead reagent (Life Technology #L10119). After washes, cells were resuspended in 200 ?L Flow buffer. Cells were then analysed on a Cytoflex cytometer (Beckman Coulter). Data were analysed using FlowJo software (Version 10, FlowJo, LLC, Ashland, USA) gating on the live CD3+ population. The MFI values from PE channel were then calculated and plotted against concentration. Curves fitting was obtained using sigmoidal 4PL equation from GraphPad Prism software.
(221) BTN3A Surface Expression on Human and Cynomolgus Circulating Cells.
(222) For BTN3A surface expression on leukocytes, 100 ul of Cynomolgus and human whole blood were plated in 96 well plates with a cocktail of specific antibodies (Aqua live Dead reagent, CD20-V450, CD8-BV605, CD4-BV650, Vg9 TCR-FITC, anti-BTN3A-PE (clone 20.1) (or mIgG1-PE for isotype control), CD3-PeCy7, CD45-AF700 and CD14-APC-H7) and incubated 15 minutes at RT protected from light. Then, red blood cells were lysed with 900 ul of lysing reagent (BD Bioscience #349202) according to manufacturer instructions. Cells were washed and analysed using multiparametric Flow cytometry. For BTN3A surface expression on Red blood cells and platelets, 100 ul of cynomolgus or human whole blood were diluted with 100 ul of PBS and incubated with a specific cocktail of antibodies (Aqua live dead, CD41-APC, CD45-AF700 and anti-BTN3A (clone 20.1) (or mIgG1-PE for isotype control)) for 15 minutes at RT protected from light. After washes, cells were analysed using multiparametric Flow cytometry. In order to obtain a relative quantification of BTN3A surface expression, calibrating beads (CellQuant Calibrator Biocytex #7208) and goat anti-mouse IgG (H+L)-PE were used in parallel according to manufacturer instruction. For every cell subset, MFI of the PE channel was reported for anti-BTN3A and isotype control. Analysis was performed by subtracting the MFI of the isotype control from the MFI of the anti-BTN3A staining and relative surface expression was calculated based on the standard curve obtained with calibration beads.
(223) Cynomolgus V?9V?2 T Cell In-Vitro Expansion and Activation.
(224) Cynomolgus whole blood from 3 animals was treated with red blood lysis buffer. After extensive washes, leukocytes were plated at 1.5M/ml of RPMI 10% SVF in 6 well plates in presence of rHuIL-2 (200 IU/ml) and mAb1 (10 ug/ml). rHuIL-2 was added at day 6 and 8 to mimic the usual cell expansion protocol used with human PBMC, and improve V?9V?2 T cell long term in vitro survival in order to obtain sufficient number of cells to perform functional assays. The percentage of V?9+ T cells was assessed at day 0, 6, 8 and 10 using a cocktail of specific antibodies and flow cytometry analysis (CD3-PC7 BD Bioscience #557749, Vg9 TCR-FITC clone 7A5 Invitrogen #TCR2720, Live Dead Near IR ThermoFisher #L10119).
(225) At day 10, expanded V?9V?2 T cells were counted and co-culture at E:T ratio of 1:1 with human tumoral cell lines. 100 000 target tumoral cell lines (Raji, DAUDI and K562) were mixed with 100 000 cynomolgus V?9V?2 T cells in presence of mAb1 or hIgG1 isotype control (10 ug/ml) or PMA (20 ng/ml)/ionomycin (1 ug/ml) used as positive control in 96 well plates. V?9V?2 T-cell degranulation was monitored after 4 hrs using CD107a/b (BD bioscience #555800) staining and flow cytometry analysis.
(226) Cynomolgus Monkey In-Vivo Study
(227) In Life Study
(228) Four- to 6-year-old, 3- to 5-kg, cynomolgus monkeys (Macaca fascicularis) of Vietnamese origin were used in this study. All animals were maintained and used in accordance with guidelines of the institutional animal care and use committee in a GLP animal facility. As a breeder health procedure, all animals were tested for tuberculosis, and prophylactic treatments were documented in the breeder's records. After arrival, animals were acclimated to study procedures for a period of at least 2 weeks. A clinical inspection for ill-health and testing for were performed. An animal health assessment was performed by a Veterinarian before the start of the predose phase to confirm the suitability of every animal for the study.
(229) mAb1 was administered intravenously (chair restrained, infusion over 15 minutes) after disinfecting the skin of non-fasted animals. Animals of Groups 1, 4, and 5 were dosed on Days 1, 8, 15, and 22. Animals of Groups 2 and 3 were dosed once on Day 1.
(230) Pharmacokinetics
(231) A qualified pharmacokinetic assay was developed for the quantification of mAb1 in cynomolgus monkey serum. Briefly, mAb1 is quantified using ELISA by spectrophotometry. A Streptavidin precoated plate is used to capture the human IgG-Fc PK Biotin Conjugate. mAb1 is then captured on the surface of the plate and the bound analyte is detected using the Goat anti-human IgG-HRP (Fc specific) antibody, a peroxidase-labelled anti-species antibody. The target range of quantification is from 90 ng/mL to 10000 ng/mL in neat serum.
(232) Immunophenotyping
(233) Blood samples (1.0 mL) were withdrawn from all animals from the vena cephalica antebrachii or vena saphena into Li-Heparin tubes. Immunophenotyping was done with cocktails of specific monoclonal antibodies. Analyses of relative cell numbers (percentage of lymphocytes/leucocytes) were performed. Total granulocyte/lymphocyte/leucocyte counts were determined on the same day by hemoanalyser and used for calculation of absolute numbers. Absolute numbers of the lymphocyte subpopulations were computed from relative numbers.
(234) Receptor Occupancy
(235) Blood samples (400 ?L) were withdrawn from all animals from the vena cephalica antebrachii or vena saphena into Li-Heparin tubes. A labeled antibody binding non-competitively to BTN3A (clone 103.2) on cells, which was pre-incubated with a surplus of ICT01 was used to determine the total surface BTN3A expression on CD3+ T-cells and CD19+ B cells. For detection of free BTN3A binding sites, the competitive binding of unlabelled mAb1 to BTN3A which inhibits the binding of fluorescence dye labeled anti-BTN3A antibody (mAb1) on CD3+ T-cells and CD19+ B cells was used. Depending on the amount of BTN3A that was blocked by mAb1, the mean fluorescence intensity (MFI) of conjugated-mAb1 was measured as geometric mean was reduced. Consequently, a staining intensity of Ab7.2 close to that of the isotype antibody was indicative of a complete saturation.
(236) b. Results
(237) mAb1 Binds to Cynomolgus BTN3A.
(238) As differential identification of the 3 BTN3A isoform genes was not possible from public databases, ImCheck performed targeted PCR on cDNA isolated from cynomolgus PBMC, using a highly conserved cDNA sequence close to the transmembrane domain to design a relevant primer. Sequencing of PCR products allowed identification of ectodomain sequences for cynomolgus BTN3A1, BTN3A2, and BTN3A3. Recombinant ectodomains of cynomolgus BTN3A1, BTN3A2 and BTN3A3 isoforms fused to a 6?His tag were produced in CHO cells based on these sequences (SEQ ID NO 21, 22 and 23 respectively).
(239) Recombinant proteins were tested for mAb1 binding by BIAcore and ELISA (Table 26). BIAcore results showed mAb1 advantageously binds to the 3 cynomolgus recombinant BTN3A1, BTN3A2 and BTN3A3, although with a lower affinity for the BTN3A1 isoform. ELISAs were performed on BTN3A1 isoforms. Interestingly, Table 26 shows comparable EC50 for mAb1 binding on recombinant human or cynomolgus BTN3A1.
(240) TABLE-US-00025 TABLE 26: mAb1 Binding to Human and Cynomolgus BTN3A Recombinant Proteins K.sub.D (M) ? EC.sub.50 (M) ? Protein Tag BIAcore (MCK) ELISA Human BTN3A1 C-ter His 0.408E?9 0.93E?9 (Sino biological # 15973-H08H) CynoBTN3A1 C-ter-His 83.8E?09 1.33E?9 CynoBTN3A2 C-ter His 5.97E?09 nd CynoBTN3A3 C-ter His 2.67E?09 nd nd: not determined
(241) In parallel, mAb1 binding on cynomolgus PBMCs was evaluated by flow cytometry. The mean EC50 of mAb1 binding to cynomolgus CD3+ T cells was comparable to that of human CD3+ T cells (Table 27). Target expression on different immune cell subpopulations from cynomolgus versus human healthy donor whole blood was addressed by multiparameter flow cytometry, using PE-conjugated anti-BTN3A mAb (clone 20.1) together with a panel of phenotyping antibodies with known cross-reactivities for human and cynomolgus macaque cell surface markers (Data not shown). These results show that BTN3A is expressed in a broad panel of peripheral blood cell populations in both species, although an apparent, generally lower expression was observed in cynomolgus macaques.
(242) TABLE-US-00026 TABLE 27 mAb1 Binding on Human and Cynomolgus CD3+ T Cells EC.sub.50 EC.sub.50 (?g/mL) (?10.sup.-9M) Cells Origin Description Gating (+/?sem) (+/?sem) Human HV EFS Primary CD3+ 5.25 34.9 PBMCs Marseille (n = 6) (+/?0.57) (+/?3.8) Cynomolgus Covance Primary CD3+ 7.02 46.79 PBMCs Munster (n = 6) (+/?0.89) (+/?5.94)
mAb1 Promote Cynomolgus V?9V?2 T-Cell Expansion and Activation In-Vitro.
(243) Next, the functional activity of mAb1 was assessed on cynomolgus V?9V?2 T cell in vitro. First, we evaluated whether mAb1 promotes cynomolgus V?9V?2 T cell expansion when incubated for 10 days with cynomolgus PBMCs. As shown in
(244) In conclusion, the results show that (i) mAb1 binds to cynomolgus cells with a similar avidity to human cells, (ii) BTN3A is expressed on the same blood cells in humans and cynomolgus monkeys, although a lower expression level was observed in the latter species, and (iii) mAb1 promotes V?9V?2 T cell subset expansion in cynomolgus PBMC, and expanded cells are reactive against mAb1-pulsed tumour target cells.
(245) mAb1 Affects Cynomolgus Vg9Vd2 T-Cell Compartment In-Vivo.
(246) An in-vivo study was conducted in 4 to 6 years old healthy female cynomolgus monkey which received single or repeated intravenous infusions of mAb1 (Table 28).
(247) The intravenous route of administration was chosen because it is the intended human therapeutic route. Animals were treated with mAb1 according to a staggered escalating dose design.
(248) TABLE-US-00027 TABLE 28 mAb1 PK/PD/Tolerability Study Design Number Number and Dose of weekly gender of Group Test item (mg/kg) injections animals 1 mAb1 0.1-1-10-100 4 1F (ascending doses) (sentinel animal) 2 1 1 3F 3 10 1 2F 4 10 4 2F 5 100 4 2F
(249) The following endpoints were evaluated: clinical signs, bodyweights, clinical pathology (haematology, clinical chemistry and coagulation), immunophenotyping for peripheral blood leukocyte populations, activation/proliferation/differentiation markers, pharmacokinetics and pharmacodynamics (BTN3A receptor occupancy on circulating T and B cells).
(250) All animals survived up to the scheduled necropsy on Day 29 of the study, after receiving single or 4 repeated 15-minute infusions of the mAb1. No test article-related effects were found on body weights or food-consumption. Clinical signs were consistent with observations seen in cynomolgus monkeys in laboratory housing settings and thus, not attributable to a test article.
(251) Pharmacokinetics
(252) mAb1 showed approximately dose-proportional pharmacokinetic behaviour following intravenous (IV) dosing over the dose range of 0.1 to 100 mg/kg (Data not shown), and a long elimination half-life typical of IgG mAbs in the absence of target-mediated clearance. Following repeated doses of 10 or 100 mg/kg/week for 4 doses (Data not shown), exposure was maintained in all animals over the treatment period, with only minimal accumulation over the 4-week treatment period. The pharmacokinetic profiles for the sentinel animal following the doses of 1 and 10 mg/kg IV in an escalating weekly dosing regimen showed some evidence for increased clearance towards the end of the weekly dosing interval which may be a result of formation of ADAs to mAb1; there was no evidence for increased clearance after the last IV dose of 100 mg/kg.
(253) Receptor Occupancy (RO)
(254) According to the BTN3A expression profile and cell population representation in blood, mAb1 RO was measured on CD3+ T cells and CD20+ B cells.
(255) The results show that BTN3A is rapidly occupied after mAb1 injection on both CD3+ T cells and CD20+ B cells (Data not shown). Repeated dosing at 100 mg/kg mAb1 appeared to be required for a full receptor occupancy throughout the weekly dosing interval.
(256) Immunophenotyping
(257) At selected time-points after each dose, blood of animals receiving mAb1 has been stained with a cocktail of specific mAbs to quantify T cells subsets (CD4, CD8, V?9 T cells, regulatory T cells), B cells, monocytes, NK cells, mDCs, pDCs and granulocytes and associated activation markers (CD69, CD86, CD95, Granzyme B, Ki67) and analysed by flow cytometry. Analysis included relative cell numbers (percentage of lymphocytes/leucocytes) for each population together with absolute cell numbers extrapolated from total lymphocyte/leucocyte counts determined from blood samples harvested at same time and analysed using an haematological cell counter.
(258) The main observations from this broad analysis are:
(259) V?9+ T cells (% among CD3+) significantly drop after dosing in all animals receiving mAb1 and come back up progressively on single dose animals. This effect appears to be specific, as it is not observed in CD4 and CD8 ?? T cells, and is suggestive of ?? T cell activation and margination on tissues, as observed for CD3 engager bi-specifics antibodies in monkeys and humans (Smith et al., 2015 Sci Rep. 2015 Dec. 11; 5:17943. doi: 10.1038/srep17943.). The results are shown in
(260) This study shows that mAb1, when administered by the IV route, appears to be well tolerated at doses up to 100 mg/kg/week. Moreover, among T cell subsets, V?9V?2 T cells are specifically and significantly affected by mAb1.
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